Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

PubMed Central

Hong, Mei; Su, Yongchao

2011-01-01

Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

Increased curvature of hollow fiber membranes could up-regulate differential functions of renal tubular cell layers.

PubMed

Shen, Chong; Meng, Qin; Zhang, Guoliang

2013-08-01

Tissue engineering devices as in vitro cell culture systems in scaffolds has encountered the bottleneck due to their much lower cell functions than real tissues/organs in vivo. Such situation has been improved in some extent by mimicking the cell microenvironments in vivo from either chemical or physical ways. However, microenvironmental curvature, commonly seen in real tissues/organs, has never been manipulated to regulate the cell performance in vitro. In this regard, this paper fabricated polysulfone membranes with or without polyethylene glycol modification to investigate the impact of curvature on two renal tubular cells. Regardless the varying membrane curvatures among hollow fiber membranes of different diameters and flat membrane of zero curvature, both renal cells could well attach at 4 h of seeding and form similar confluent layers at 6 days on each membrane. Nevertheless, the renal cells on hollow fibers, though showing confluent morphology as those on flat membranes, expressed higher renal functions and, moreover, the renal functions significantly increased with the membrane curvature among hollow fibers. Such upregulation on functions was unassociated with mass transport barrier of hollow fibers, because the cultures on lengthwise cut hollow fibers without mass transfer barrier showed same curvature effect on renal functions as whole hollow fibers. It could be proposed that the curvature of hollow fiber membrane approaching to the large curvature in kidney tubules increased the mechanical stress in the renal cells and thus might up-regulate the renal cell functions. In conclusion, the increase of substrate curvature could up-regulate the cell functions without altering the confluent cell morphology and this finding will facilitate the design of functional tissue engineering devices. Copyright © 2013 Wiley Periodicals, Inc.

Micro pore arrays in free standing cyclic olefin copolymer membranes: fabrication and surface functionalization strategies for in-vitro barrier tissue models

NASA Astrophysics Data System (ADS)

Gel, M.; Kandasamy, S.; Cartledge, K.; Be, C. L.; Haylock, D.

2013-12-01

In recent years there has been growing interest in micro engineered in-vitro models of tissues and organs. These models are designed to mimic the in-vivo like physiological conditions with a goal to study human physiology in an organ-specific context or to develop in-vitro disease models. One of the challenges in the development of these models is the formation of barrier tissues in which the permeability is controlled locally by the tissues cultured at the interface. In-vitro models of barrier tissues are typically created by generating a monolayer of cells grown on thin porous membranes. This paper reports a robust preparation method for free standing porous cyclic olefin copolymer (COC) membranes. We also demonstrate that gelatin coated membranes facilitate formation of highly confluent monolayer of HUVECs. Membranes with thickness in the range of 2-3 um incorporating micro pores with diameter approximately 20 um were fabricated and integrated with microfluidic channels. The performance of the device was demonstrated with a model system mimicking the endothelial barrier in bone marrow sinusoids.

Direct visualization of the arterial wall water permeability barrier using CARS microscopy

PubMed Central

Lucotte, Bertrand M.; Powell, Chloe; Knutson, Jay R.; Combs, Christian A.; Malide, Daniela; Yu, Zu-Xi; Knepper, Mark; Patel, Keval D.; Pielach, Anna; Johnson, Errin; Borysova, Lyudmyla; Balaban, Robert S.

2017-01-01

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies. PMID:28373558

Ultrathin Ceramic Membranes as Scaffolds for Functional Cell Coculture Models on a Biomimetic Scale

PubMed Central

Jud, Corinne; Ahmed, Sher; Müller, Loretta; Kinnear, Calum; Vanhecke, Dimitri; Umehara, Yuki; Frey, Sabine; Liley, Martha; Angeloni, Silvia; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Abstract Epithelial tissue serves as an interface between biological compartments. Many in vitro epithelial cell models have been developed as an alternative to animal experiments to answer a range of research questions. These in vitro models are grown on permeable two-chamber systems; however, commercially available, polymer-based cell culture inserts are around 10 μm thick. Since the basement membrane found in biological systems is usually less than 1 μm thick, the 10-fold thickness of cell culture inserts is a major limitation in the establishment of realistic models. In this work, an alternative insert, accommodating an ultrathin ceramic membrane with a thickness of only 500 nm (i.e., the Silicon nitride Microporous Permeable Insert [SIMPLI]-well), was produced and used to refine an established human alveolar barrier coculture model by both replacing the conventional inserts with the SIMPLI-well and completing it with endothelial cells. The structural–functional relationship of the model was evaluated, including the translocation of gold nanoparticles across the barrier, revealing a higher translocation if compared to corresponding polyethylene terephthalate (PET) membranes. This study demonstrates the power of the SIMPLI-well system as a scaffold for epithelial tissue cell models on a truly biomimetic scale, allowing construction of more functionally accurate models of human biological barriers. PMID:26713225

Physiological and pathophysiological factors affecting the expression and activity of the drug transporter MRP2 in intestine. Impact on its function as membrane barrier.

PubMed

Arana, Maite R; Tocchetti, Guillermo N; Rigalli, Juan P; Mottino, Aldo D; Villanueva, Silvina S M

2016-07-01

The gastrointestinal epithelium functions as a selective barrier to absorb nutrients, electrolytes and water, but at the same time restricts the passage into the systemic circulation of intraluminal potentially toxic compounds. This epithelium maintains its selective barrier function through the presence of very selective and complex intercellular junctions and the ability of the absorptive cells to reject those compounds. Accordingly, the enterocytes metabolize orally incorporated xenobiotics and secrete the hydrophilic metabolites back into the intestinal lumen through specific transporters localized apically. In the recent decades, there has been increasing recognition of the existence of the intestinal cellular barrier. In the present review we focus on the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) in the apical membrane of the enterocytes, as an important component of this intestinal barrier, as well as on its regulation. We provide a detailed compilation of significant contributions demonstrating that MRP2 expression and function vary under relevant physiological and pathophysiological conditions. Because MRP2 activity modulates the availability and pharmacokinetics of many therapeutic drugs administered orally, their therapeutic efficacy and safety may vary as well. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rupturing the hemi-fission intermediate in membrane fission under tension: Reaction coordinates, kinetic pathways, and free-energy barriers

NASA Astrophysics Data System (ADS)

Zhang, Guojie; Müller, Marcus

2017-08-01

Membrane fission is a fundamental process in cells, involved inter alia in endocytosis, intracellular trafficking, and virus infection. Its underlying molecular mechanism, however, is only incompletely understood. Recently, experiments and computer simulation studies have revealed that dynamin-mediated membrane fission is a two-step process that proceeds via a metastable hemi-fission intermediate (or wormlike micelle) formed by dynamin's constriction. Importantly, this hemi-fission intermediate is remarkably metastable, i.e., its subsequent rupture that completes the fission process does not occur spontaneously but requires additional, external effects, e.g., dynamin's (unknown) conformational changes or membrane tension. Using simulations of a coarse-grained, implicit-solvent model of lipid membranes, we investigate the molecular mechanism of rupturing the hemi-fission intermediate, such as its pathway, the concomitant transition states, and barriers, as well as the role of membrane tension. The membrane tension is controlled by the chemical potential of the lipids, and the free-energy landscape as a function of two reaction coordinates is obtained by grand canonical Wang-Landau sampling. Our results show that, in the course of rupturing, the hemi-fission intermediate undergoes a "thinning → local pinching → rupture/fission" pathway, with a bottle-neck-shaped cylindrical micelle as a transition state. Although an increase of membrane tension facilitates the fission process by reducing the corresponding free-energy barrier, for biologically relevant tensions, the free-energy barriers still significantly exceed the thermal energy scale kBT.

Rupturing the hemi-fission intermediate in membrane fission under tension: Reaction coordinates, kinetic pathways, and free-energy barriers.

PubMed

Zhang, Guojie; Müller, Marcus

2017-08-14

Membrane fission is a fundamental process in cells, involved inter alia in endocytosis, intracellular trafficking, and virus infection. Its underlying molecular mechanism, however, is only incompletely understood. Recently, experiments and computer simulation studies have revealed that dynamin-mediated membrane fission is a two-step process that proceeds via a metastable hemi-fission intermediate (or wormlike micelle) formed by dynamin's constriction. Importantly, this hemi-fission intermediate is remarkably metastable, i.e., its subsequent rupture that completes the fission process does not occur spontaneously but requires additional, external effects, e.g., dynamin's (unknown) conformational changes or membrane tension. Using simulations of a coarse-grained, implicit-solvent model of lipid membranes, we investigate the molecular mechanism of rupturing the hemi-fission intermediate, such as its pathway, the concomitant transition states, and barriers, as well as the role of membrane tension. The membrane tension is controlled by the chemical potential of the lipids, and the free-energy landscape as a function of two reaction coordinates is obtained by grand canonical Wang-Landau sampling. Our results show that, in the course of rupturing, the hemi-fission intermediate undergoes a "thinning → local pinching → rupture/fission" pathway, with a bottle-neck-shaped cylindrical micelle as a transition state. Although an increase of membrane tension facilitates the fission process by reducing the corresponding free-energy barrier, for biologically relevant tensions, the free-energy barriers still significantly exceed the thermal energy scale k B T.

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity*

PubMed Central

Peters, Diane E.; Szabo, Roman; Friis, Stine; Shylo, Natalia A.; Uzzun Sales, Katiuchia; Holmbeck, Kenn; Bugge, Thomas H.

2014-01-01

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat−/Cat− mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8−/− and Prss8Cat−/Cat− mice born within the same litter. Furthermore, Prss8Cat−/Cat− mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease. PMID:24706745

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PubMed

Ban, Yuriko; Cooper, Leanne J; Fullwood, Nigel J; Nakamura, Takahiro; Tsuzuki, Masakatsu; Koizumi, Noriko; Dota, Atsuyoshi; Mochida, Chikako; Kinoshita, Shigeru

2003-06-01

To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

Nanoscopic compartmentalization of membrane protein motion at the axon initial segment.

PubMed

Albrecht, David; Winterflood, Christian M; Sadeghi, Mohsen; Tschager, Thomas; Noé, Frank; Ewers, Helge

2016-10-10

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal, and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axonal and somatodendritic domains. Recently, an axonal periodic pattern of actin, spectrin, and ankyrin forming 190-nm-spaced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear. Here, we performed single-particle tracking time-course experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single-particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton. We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ∼190-nm-spaced segments along the AIS axis as early as day in vitro 4, and this pattern alternates with actin rings. Mathematical modeling shows that diffusion barriers between the segments significantly reduce lateral diffusion along the axon. © 2016 Albrecht et al.

Placenta-on-a-chip: a novel platform to study the biology of the human placenta.

PubMed

Lee, Ji Soo; Romero, Roberto; Han, Yu Mi; Kim, Hee Chan; Kim, Chong Jai; Hong, Joon-Seok; Huh, Dongeun

2016-01-01

Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta. A "Placenta-on-a-Chip" microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone. Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations. The "Placenta-on-a-Chip" microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.

Membrane curvature and its generation by BAR proteins

PubMed Central

Mim, Carsten; Unger, Vinzenz M

2012-01-01

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent. PMID:23058040

Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

PubMed

Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

2016-02-01

A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

THE ANATOMIC SITE OF THE TRANSEPITHELIAL PERMEABILITY BARRIERS OF TOAD BLADDER

PubMed Central

DiBona, Donald R.; Civan, Mortimer M.; Leaf, Alexander

1969-01-01

An examination of the mucosal epithelium of the urinary bladder of the toad reveals that the two major cell types which abut on the urinary surface, the granular and mitochondria-rich cells, also contact the basement membrane. Thus, the epithelium functions as a single cell layer. Although basal cells are interpolated between the granular cells and the basement membrane over a large portion of the epithelium, they do not constitute an additional continuous cell layer. This finding is consistent with extensive physiological data which had assumed that the major permeability barriers of this epithelium were the apical and basal-lateral plasma membranes of a single layer of cells. PMID:5782445

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

PubMed

Liévin-Le Moal, Vanessa

2013-06-01

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier. © 2013 John Wiley & Sons Ltd.

Fatty acids are required for epidermal permeability barrier function.

PubMed

Mao-Qiang, M; Elias, P M; Feingold, K R

1993-08-01

The permeability barrier is mediated by a mixture of ceramides, sterols, and free fatty acids arranged as extracellular lamellar bilayers in the stratum corneum. Whereas prior studies have shown that cholesterol and ceramides are required for normal barrier function, definitive evidence for the importance of nonessential fatty acids is not available. To determine whether epidermal fatty acid synthesis also is required for barrier homeostasis, we applied 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an inhibitor of acetyl CoA carboxylase, after disruption of the barrier by acetone or tape stripping. TOFA inhibits epidermal fatty acid by approximately 50% and significantly delays barrier recovery. Moreover, coadministration of palmitate with TOFA normalizes barrier recovery, indicating that the delay is due to a deficiency in bulk fatty acids. Furthermore, TOFA treatment also delays the return of lipids to the stratum corneum and results in abnormalities in the structure of lamellar bodies, the organelle which delivers lipid to the stratum corneum. In addition, the organization of secreted lamellar body material into lamellar bilayers within the stratum corneum interstices is disrupted by TOFA treatment. Finally, these abnormalities in lamellar body and stratum corneum membrane structure are corrected by coapplication of palmitate with TOFA. These results demonstrate a requirement for bulk fatty acids in barrier homeostasis. Thus, inhibiting the epidermal synthesis of any of the three key lipids that form the extracellular, lipid-enriched membranes of the stratum corneum results in an impairment in barrier homeostasis.

Fatty acids are required for epidermal permeability barrier function.

PubMed Central

Mao-Qiang, M; Elias, P M; Feingold, K R

1993-01-01

The permeability barrier is mediated by a mixture of ceramides, sterols, and free fatty acids arranged as extracellular lamellar bilayers in the stratum corneum. Whereas prior studies have shown that cholesterol and ceramides are required for normal barrier function, definitive evidence for the importance of nonessential fatty acids is not available. To determine whether epidermal fatty acid synthesis also is required for barrier homeostasis, we applied 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an inhibitor of acetyl CoA carboxylase, after disruption of the barrier by acetone or tape stripping. TOFA inhibits epidermal fatty acid by approximately 50% and significantly delays barrier recovery. Moreover, coadministration of palmitate with TOFA normalizes barrier recovery, indicating that the delay is due to a deficiency in bulk fatty acids. Furthermore, TOFA treatment also delays the return of lipids to the stratum corneum and results in abnormalities in the structure of lamellar bodies, the organelle which delivers lipid to the stratum corneum. In addition, the organization of secreted lamellar body material into lamellar bilayers within the stratum corneum interstices is disrupted by TOFA treatment. Finally, these abnormalities in lamellar body and stratum corneum membrane structure are corrected by coapplication of palmitate with TOFA. These results demonstrate a requirement for bulk fatty acids in barrier homeostasis. Thus, inhibiting the epidermal synthesis of any of the three key lipids that form the extracellular, lipid-enriched membranes of the stratum corneum results in an impairment in barrier homeostasis. Images PMID:8102380

Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors

PubMed Central

2012-01-01

Background Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out. Results In ischemia-damaged pig jejunum, using measurements of transepithelial resistance, 3H-mannitol fluxes, short-circuit current (Cl− secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 μM linaclotide and 1 μM lubiprostone both caused similar increases in short-circuit current (Cl− secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca2+]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear. Conclusions Considering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS. PMID:22553939

Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors.

PubMed

Cuppoletti, John; Blikslager, Anthony T; Chakrabarti, Jayati; Nighot, Prashant K; Malinowska, Danuta H

2012-05-03

Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out. In ischemia-damaged pig jejunum, using measurements of transepithelial resistance, (3)H-mannitol fluxes, short-circuit current (Cl(-) secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 μM linaclotide and 1 μM lubiprostone both caused similar increases in short-circuit current (Cl(-) secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear. Considering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Cell Membrane Transport Mechanisms: Ion Channels and Electrical Properties of Cell Membranes.

PubMed

Kulbacka, Julita; Choromańska, Anna; Rossowska, Joanna; Weżgowiec, Joanna; Saczko, Jolanta; Rols, Marie-Pierre

2017-01-01

Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.

The plasma membrane as a capacitor for energy and metabolism.

PubMed

Ray, Supriyo; Kassan, Adam; Busija, Anna R; Rangamani, Padmini; Patel, Hemal H

2016-02-01

When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as "capacitors for energy and metabolism." Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell.

Oblique wave trapping by vertical permeable membrane barriers located near a wall

NASA Astrophysics Data System (ADS)

Koley, Santanu; Sahoo, Trilochan

2017-12-01

The effectiveness of a vertical partial flexible porous membrane wave barrier located near a rigid vertical impermeable seawall for trapping obliquely incident surface gravity waves are analyzed in water of uniform depth under the assumption of linear water wave theory and small amplitude membrane barrier response. From the general formulation of the submerged membrane barrier, results for bottom-standing and surface-piercing barriers are computed and analyzed in special cases. Using the eigenfunction expansion method, the boundary-value problems are converted into series relations and then the required unknowns are obtained using the least squares approximation method. Various physical quantities of interests like reflection coefficient, wave energy dissipation, wave forces acting on the membrane barrier and the seawall are computed and analyzed for different values of the wave and structural parameters. The study will be useful in the design of the membrane wave barrier for the creation of tranquility zone in the lee side of the barrier to protect the seawall.

Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers

NASA Astrophysics Data System (ADS)

Lewis, Shanta

Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4microg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4microg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.

Tight junction-based epithelial microenvironment and cell proliferation.

PubMed

Tsukita, S; Yamazaki, Y; Katsuno, T; Tamura, A; Tsukita, S

2008-11-24

Belt-like tight junctions (TJs), referred to as zonula occludens, have long been regarded as a specialized differentiation of epithelial cell membranes. They are required for cell adhesion and paracellular barrier functions, and are now thought to be partly involved in fence functions and in cell polarization. Recently, the molecular bases of TJs have gradually been unveiled. TJs are constructed by TJ strands, whose basic frameworks are composed of integral membrane proteins with four transmembrane domains, designated claudins. The claudin family is supposedly composed of at least 24 members in mice and humans. Other types of integral membrane proteins with four transmembrane domains, namely occludin and tricellulin, as well as the single transmembrane proteins, JAMs (junctional adhesion molecules) and CAR (coxsackie and adenovirus receptor), are associated with TJ strands, and the high-level organization of TJ strands is likely to be established by membrane-anchored scaffolding proteins, such as ZO-1/2. Recent functional analyses of claudins in cell cultures and in mice have suggested that claudin-based TJs may have pivotal functions in the regulation of the epithelial microenvironment, which is critical for various biological functions such as control of cell proliferation. These represent the dawn of 'Barriology' (defined by Shoichiro Tsukita as the science of barriers in multicellular organisms). Taken together with recent reports regarding changes in claudin expression levels, understanding the regulation of the TJ-based microenvironment system will provide new insights into the regulation of polarization in the respect of epithelial microenvironment system and new viewpoints for developing anticancer strategies.

Extracorporeal membrane oxygenation causes loss of intestinal epithelial barrier in the newborn piglet.

PubMed

Kurundkar, Ashish R; Killingsworth, Cheryl R; McIlwain, R Britt; Timpa, Joseph G; Hartman, Yolanda E; He, Dongning; Karnatak, Rajendra K; Neel, Mary L; Clancy, John P; Anantharamaiah, G M; Maheshwari, Akhil

2010-08-01

Extracorporeal membrane oxygenation (ECMO) is an important life-support system used in neonates and young children with intractable cardiorespiratory failure. In this study, we used our porcine neonatal model of venoarterial ECMO to investigate whether ECMO causes gut barrier dysfunction. We subjected 3-wk-old previously healthy piglets to venoarterial ECMO for up to 8 h and evaluated gut mucosal permeability, bacterial translocation, plasma levels of bacterial products, and ultrastructural changes in gut epithelium. We also measured plasma lipopolysaccharide (LPS) levels in a small cohort of human neonates receiving ECMO. In our porcine model, ECMO caused a rapid increase in gut mucosal permeability within the first 2 h of treatment, leading to a 6- to 10-fold rise in circulating bacterial products. These changes in barrier function were associated with cytoskeletal condensation in epithelial cells, which was explained by phosphorylation of a myosin II regulatory light chain. In support of these findings, we also detected elevated plasma LPS levels in human neonates receiving ECMO, indicating a similar loss of gut barrier function in these infants. On the basis of these data, we conclude that ECMO is an independent cause of gut barrier dysfunction and bacterial translocation may be an important contributor to ECMO-related inflammation.

Extracorporeal Membrane Oxygenation Causes Loss of Intestinal Epithelial Barrier in the Newborn Piglet

PubMed Central

Kurundkar, Ashish R.; Killingsworth, Cheryl R.; McILwain, R. Britt; Timpa, Joseph G.; Hartman, Yolanda E.; He, Dongning; Karnatak, Rajendra K.; Neel, Mary Lauren; Clancy, John P.; Anantharamaiah, G. M.; Maheshwari, Akhil

2010-01-01

Extracorporeal membrane oxygenation (ECMO) is an important life-support system used in neonates and young children with intractable cardiorespiratory failure. In this study, we used our porcine neonatal model of venoarterial ECMO to investigate whether ECMO causes gut barrier dysfunction. We subjected 3-week-old previously-healthy piglets to venoarterial ECMO for up to 8 hours and evaluated gut mucosal permeability, bacterial translocation, plasma levels of bacterial products, and ultrastructural changes in gut epithelium. We also measured plasma lipopolysaccharide (LPS) levels in a small cohort of human neonates receiving ECMO. In our porcine model, ECMO caused a rapid increase in gut mucosal permeability within the first 2 hours of treatment, leading to a 6–10 fold rise in circulating bacterial products. These changes in barrier function were associated with cytoskeletal condensation in epithelial cells, which was explained by phosphorylation of a myosin II regulatory light chain. In support of these findings, we also detected elevated plasma LPS levels in human neonates receiving ECMO, indicating a similar loss of gut barrier function in these infants. Based on these data, we conclude that ECMO is an independent cause of gut barrier dysfunction, and that bacterial translocation may be an important contributor to ECMO-related inflammation. PMID:20442689

31P and 1H NMR Studies of the Molecular Organization of Lipids in the Parallel Artificial Membrane Permeability Assay.

PubMed

Assmus, Frauke; Ross, Alfred; Fischer, Holger; Seelig, Joachim; Seelig, Anna

2017-01-03

The parallel artificial membrane permeability assay (PAMPA) has emerged as a widely used primary in vitro screen for passive permeability of potential drug candidates. However, the molecular structure of the permeation barrier (consisting of a filter-supported dodecane-egg lecithin mixture) has never been characterized. Here, we investigated the long-range order of phospholipids in the PAMPA barrier by means of 31 P static solid-state NMR. Diffusion constants of PAMPA membrane components were derived from liquid state NMR and, in addition, drug distribution between the PAMPA lipid phase and buffer (log D PAMPA at pH 7.4) was systematically investigated. Increasing concentration of n-dodecane to the system egg lecithin-water (lamellar phase, L α ) induces formation of inverted hexagonal (H ii ) and isotropic phases. At n-dodecane concentrations matching those used in PAMPA (9%, w/v) a purely "isotropic" phase was observed corresponding to lipid aggregates with a diameter in the range 4-7 nm. Drug distribution studies indicate that these reverse micelles facilitate the binding to, and in turn the permeation across, the PAMPA dodecane barrier, in particular for amphiphilic solutes. The proposed model for the molecular architecture and function of the PAMPA barrier provides a fundamental, hitherto missing framework to evaluate the scope but also limitations of PAMPA for the prediction of in vivo membrane permeability.

Protein adsorption capability on polyurethane and modified-polyurethane membrane for periodontal guided tissue regeneration applications.

PubMed

Sheikh, Zeeshan; Khan, Abdul Samad; Roohpour, Nima; Glogauer, Michael; Rehman, Ihtesham U

2016-11-01

Periodontal disease if left untreated can result in creation of defects within the alveolar ridge. Barrier membranes are frequently used with or without bone replacement graft materials for achieving periodontal guided tissue regeneration (GTR). Surface properties of barrier membranes play a vital role in their functionality and clinical success. In this study polyetherurethane (PEU) membranes were synthesized by using 4,4'-methylene-diphenyl diisocyanate (MDI), polytetramethylene oxide (PTMO) and 1,4-butane diol (BDO) as a chain extender via solution polymerization. Hydroxyl terminated polydimethylsiloxane (PDMS) due to having inherent surface orientation towards air was used for surface modification of PEU on one side of the membranes. This resulting membranes had one surface being PEU and the other being PDMS coated PEU. The prepared membranes were treated with solutions of bovine serum albumin (BSA) in de-ionized water at 37°C at a pH of 7.2. The surface protein adsorptive potential of PEU membranes was observed using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Raman spectroscopy and Confocal Raman spectroscopy. The contact angle measurement, tensile strength and modulus of prepared membranes were also evaluated. PEU membrane (89.86±1.62°) exhibited less hydrophobic behavior than PEU-PDMS (105.87±3.16°). The ultimate tensile strength and elastic modulus of PEU (27±1MPa and 14±2MPa) and PEU-PDMS (8±1MPa and 26±1MPa) membranes was in required range. The spectral analysis revealed adsorption of BSA proteins on the surface of non PDMS coated PEU surface. The PDMS modified PEU membranes demonstrated a lack of BSA adsorption. The non PDMS coated side of the membrane which adsorbs proteins could potentially be used facing towards the defect attracting growth factors for periodontal tissue regeneration. Whereas, the PDMS coated side could serve as an occlusive barrier for preventing gingival epithelial cells from proliferating and migrating into the defect space by facing the soft tissue flaps. This study demonstrates the potential of a dual natured PEU barrier membrane for use in periodontal tissue engineering applications and further investigations are required. Copyright © 2016 Elsevier B.V. All rights reserved.

A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics

PubMed Central

Delgado, Fernanda; Umans, Benjamin D.; Gerding, Matthew A.; Davis, Brigid M.

2016-01-01

Gram-negative bacteria are notoriously resistant to a variety of high-molecular-weight antibiotics due to the limited permeability of their outer membrane (OM). The basis of OM barrier function and the genetic factors required for its maintenance remain incompletely understood. Here, we employed transposon insertion sequencing to identify genes required for Vibrio cholerae resistance to vancomycin and bacitracin, antibiotics that are thought to be too large to efficiently penetrate the OM. The screen yielded several genes whose protein products are predicted to participate in processes important for OM barrier functions and for biofilm formation. In addition, we identified a novel factor, designated vigA (for vancomycin inhibits growth), that has not previously been characterized or linked to outer membrane function. The vigA open reading frame (ORF) codes for an inner membrane protein, and in its absence, cells became highly sensitive to glycopeptide antibiotics (vancomycin and ramoplanin) and bacitracin but not to other large antibiotics or detergents. In contrast to wild-type (WT) cells, the vigA mutant was stained with fluorescent vancomycin. These observations suggest that VigA specifically prevents the periplasmic accumulation of certain large antibiotics without exerting a general role in the maintenance of OM integrity. We also observed marked interspecies variability in the susceptibilities of Gram-negative pathogens to glycopeptides and bacitracin. Collectively, our findings suggest that the OM barrier is not absolute but rather depends on specific OM-antibiotic interactions. PMID:27216069

The plasma membrane as a capacitor for energy and metabolism

PubMed Central

Ray, Supriyo; Kassan, Adam; Busija, Anna R.; Rangamani, Padmini

2016-01-01

When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as “capacitors for energy and metabolism.” Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell. PMID:26771520

Structural and functional maturation of rat gastrointestinal barrier with thyroxine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Israel, E.J.; Pang, K.Y.; Harmatz, P.R.

It has been noted that the closure of the intestinal barrier to immunoglobulins is a normal maturational process in the rat. It has also been noted that the microvillus membrane (MVM) of newborn animals differs from adult MVM. The purpose of this study is to document whether thyroid hormone can induce closure in vivo in the rat and to relate this effect of thyroxine to the structural and functional maturation of the intestinal MVM. To assess closure, 2-wk-old rats were fed in rat immunoglobulin G (IgG), and serum antibody binding activity was measured 4 h later. The antibody binding activitymore » of treated animals (T) was 1.5-2 times less than that of controls (C), indicating that thyroxine stimulates closure. The MVM similarly showed signs of maturation. Structural maturation was demonstrated by the lower fluidity of the thyroid-treated animals' membranes. Under the influence of thyroxine, the number of receptors on the MVM for IgG had decreased, while the K/sub a/ remained the same, demonstrating the functional maturation of the MVM. In conclusion, thryoid hormone can induce both structural and functional maturation of the intestinal MVM and can enhance the intestinal mucosal barrier by decreasing the penetration of antibodies.« less

Development of a novel multi-functional active membrane capping barrier for the remediation of nitrobenzene-contaminated sediment.

PubMed

Wang, Qing; Li, Yi; Wang, Chao; Wu, Yue; Wang, Peifang

2014-07-15

A novel bio-reactive capping barrier composed of polysulfone/granular activated carbon (PS/GAC) hybrid membranes immobilized with microorganism was developed for the remediation of nitrobenzene in sediments. The SEM observation demonstrated that all the membranes had a dense top layer and a porous sublayer, this structure can block the transfer of nitrobenzene from sediment to the water and enhance nitrobenzene degradation. Adsorption behaviors of nitrobenzene on membranes showed that the membrane impregnated with GAC had better performance than the pure PS membrane. The values of Kads increased from 4.64 (without GAC) to 6.19 (1:2 GAC). 20mg/L nitrobenzene can be completely degraded by Pseudomonas putida immobilized on membranes. The biodegradation rate of activated carbon-filled membrane system was little higher than that of pure PS membrane system. For remediation experiments, only about 21.7, 28.3 and 43.9% of nitrobenzene in the sediment was removed by the end of the experiments for PS/GAC membrane, sand-alone and sand amended with activated carbon capping systems, respectively. While for PS/GAC+microorganisms capping system, more than 70% of nitrobenzene loss was observed. This demonstrated that nitrobenzene can be effectively removed from contaminated sediments by microbial degradation in the bio-reactive capping system. Copyright © 2014 Elsevier B.V. All rights reserved.

Improving the performance of water desalination through ultra-permeable functionalized nanoporous graphene oxide membrane

NASA Astrophysics Data System (ADS)

Hosseini, Mostafa; Azamat, Jafar; Erfan-Niya, Hamid

2018-01-01

Molecular dynamics simulations were performed to investigate the water desalination performance of nanoporous graphene oxide (NPGO) membranes. The simulated systems consist of a NPGO as a membrane with a functionalized pore in its center immersed in an aqueous ionic solution and a graphene sheet as a barrier. The considered NPGO membranes are involved four types of pore with different size and chemistry. The results indicated that the NPGO membrane has effective efficiency in salt rejection as well as high performance in water flux. For all types of pore with the radius size of 2.9-4.5 Å, the NPGO shows salt rejection of >89%. Functional groups on the surface and edge of pores have a great effect on water flux. To precisely understand the effect of functional groups on the surface of nanostructured membranes, nanoporous graphene was simulated under the same condition for comparison. Hydrophilic groups on the surface make the NPGO as an ultra-permeable membrane. As a result, the obtained water flux for NPGO was about 77% greater than graphene. Also, it was found that the water flux of NPGO is 2-5 orders of magnitude greater than other existing reverse osmosis membranes. Therefore, the investigated systems can be recommended as a model for the water desalination.

Direct simulation of amphiphilic nanoparticle mediated membrane interactions

NASA Astrophysics Data System (ADS)

Tahir, Mukarram; Alexander-Katz, Alfredo

Membrane fusion is a critical step in the transport of biological cargo through membrane-bound compartments like vesicles. Membrane proteins that alleviate energy barriers for initial stalk formation and eventual rupture of the hemifusion intermediate during fusion generally assist this process. Gold nanoparticles functionalized with a combination of hydrophobic and hydrophilic alkanethiol ligands have recently been shown to induce membrane re-arrangements that are similar to those associated with these fusion proteins. In this work, we utilize molecular dynamics simulation to systematically design nanoparticles that exhibit targeted interactions with membranes. We introduce a method for rapidly parameterizing nanoparticle topology for the MARTINI biomolecular force field to permit long timescale simulation of their interactions with lipid bilayers. We leverage this model to investigate how ligand chemistry governs the nanoparticle's insertion efficacy and the perturbations it generates in the membrane environment. We further demonstrate through unbiased simulations that these nanoparticles can direct the fusion of lipid assemblies such as micelles and vesicles in a manner that mimics the function of biological fusion peptides and SNARE proteins.

Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

PubMed

Rebaud, Samuel; Maniti, Ofelia; Girard-Egrot, Agnès P

2014-12-01

Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Reactive Membrane Barriers for Containment of Subsurface Contamination

DOE Office of Scientific and Technical Information (OSTI.GOV)

William A. Arnold; Edward L. Cussler

2007-02-26

The overall goal of this project was to develop reactive membrane barriers--a new and flexible technique to contain and stabilize subsurface contaminants. Polymer membranes will leak once a contaminant is able to diffuse through the membrane. By incorporating a reactive material in the polymer, however, the contaminant is degraded or immobilized within the membrane. These processes increase the time for contaminants to breakthrough the barrier (i.e. the lag time) and can dramatically extend barrier lifetimes. In this work, reactive barrier membranes containing zero-valent iron (Fe{sup 0}) or crystalline silicotitanate (CST) were developed to prevent the migration of chlorinated solvents andmore » cesium-137, respectively. These studies were complemented by the development of models quantifying the leakage/kill time of reactive membranes and describing the behavior of products produced via the reactions within the membranes. First, poly(vinyl alcohol) (PVA) membranes containing Fe{sup 0} and CST were prepared and tested. Although PVA is not useful in practical applications, it allows experiments to be performed rapidly and the results to be compared to theory. For copper ions (Cu{sup 2+}) and carbon tetrachloride, the barrier was effective, increasing the time to breakthrough over 300 times. Even better performance was expected, and the percentage of the iron used in the reaction with the contaminants was determined. For cesium, the CST laden membranes increased lag times more than 30 times, and performed better than theoretical predictions. A modified theory was developed for ion exchangers in reactive membranes to explain this result. With the PVA membranes, the effect of a groundwater matrix on barrier performance was tested. Using Hanford groundwater, the performance of Fe{sup 0} barriers decreased compared to solutions containing a pH buffer and high levels of chloride (both of which promote iron reactivity). For the CST bearing membrane, performance improved by a factor of three when groundwater was used in place of deionized water. The performance of high density polyethylene (HDPE) membranes containing Fe{sup 0} was then evaluating using carbon tetrachloride as the target contaminant. Only with a hydrophilic additive (glycerol), was the iron able to extend lag times. Lag times were increased by a factor of 15, but only 2-3% of the iron was used, likely due to formation of oxide precipitates on the iron surface, which slowed the reaction. With thicker membranes and lower carbon tetrachloride concentrations, it is expected that performance will improve. Previous models for reactive membranes were also extended. The lag time is a measurement of when the barrier is breached, but contaminants do slowly leak through prior to the lag time. Thus, two parameters, the leakage and the kill time, were developed to determine when a certain amount of pollutant has escaped (the kill time) or when a given exposure (concentration x time) occurs (the leakage). Finally, a model was developed to explain the behavior of mobile reaction products in reactive barrier membranes. Although the goal of the technology is to avoid such products, it is important to be able to predict how these products will behave. Interestingly, calculations show that for any mobile reaction products, one half of the mass will diffuse into the containment area and one half will escape, assuming that the volumes of the containment area and the surrounding environment are much larger than the barrier membrane. These parameters/models will aid in the effective design of barrier membranes.« less

Pore-Confined Carriers and Biomolecules in Mesoporous Silica for Biomimetic Separation and Targeting

NASA Astrophysics Data System (ADS)

Zhou, Shanshan

Selectively permeable biological membranes composed of lipophilic barriers inspire the design of biomimetic carrier-mediated membranes for aqueous solute separation. This work imparts selective permeability to lipid-filled pores of silica thin film composite membranes using carrier molecules that reside in the lipophilic self-assemblies. The lipids confined inside the pores of silica are proven to be a more effective barrier than bilayers formed on the porous surface through vesicle fusion, which is critical for quantifying the function of an immobilized carrier. The ability of a lipophilic carrier embedded in the lipid bilayer to reversibly bind the target solute and transport it through the membrane is demonstrated. Through the functionalization of the silica surface with enzymes, enzymatic catalysis and biomimetic separations can be combined on this nanostructured composite platform. The successful development of biomimetic nanocomposite membrane can provide for efficient dilute aqueous solute upgrading or separations using engineered carrier/catalyst/support systems. While the carrier-mediated biomimetic membranes hold great potential, fully understanding of the transport processes in composite synthetic membranes is essential for improve the membrane performance. Electrochemical impedance spectroscopy (EIS) technique is demonstrated to be a useful tool for characterizing the thin film pore accessibility. Furthermore, the effect of lipid bilayer preparation methods on the silica thin film (in the form of pore enveloping, pore filling) on ion transport is explored, as a lipid bilayer with high electrically insulation is essential for detecting activity of proteins or biomimetic carriers in the bilayer. This study provides insights for making better barriers on mesoporous support for carrier-mediated membrane separation process. Porous silica nanoparticles (pSNPs) with pore sizes appropriate for biomolecule loading are potential for encapsulating dsRNA within the pores to achieve effective delivery of dsRNA to insects for RNA interference (RNAi). The mobility of dsRNA in the nanopores of the pSNPs is expected to have a functional effect on delivery of dsRNA to insects. The importance of pores to a mobile dsRNA network is demonstrated by the lack of measurable mobility for both lengths of RNA on nonporous materials. In addition, when the dsRNA could not penetrate the pores, dsRNA mobility is also not measurable at the surface of the particle. Thus, the pores seem to serve as a "sink" in providing a mobile network of dsRNA on the surface of the particle. This work successfully demonstrates the loading of RNA on functionalized pSNPs and identified factors that affects RNA loading and releasing, which provides basis for the delivery of RNA-loaded silica particles in vivo.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawamoto, Shuhei; Shinoda, Wataru, E-mail: w.shinoda@apchem.nagoya-u.ac.jp; Klein, Michael L.

The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle–vesicle, vesicle–planar, and planar–planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusionmore » of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.« less

Distribution of endogenous albumin in the glomerular wall of proteinuric patients.

PubMed Central

Russo, P. A.; Bendayan, M.

1990-01-01

Glomerular proteinuria seems to be related, in part, to loss or impairment of the normal barrier function of the glomerular capillary wall. To investigate the functional properties of this barrier, endogenous albumin was revealed in the glomerular wall of proteinuric patients and compared with a nonproteinuric control by immunoelectron microscopy using the protein A-gold method. In the control biopsy, peaks of albumin accumulation were noted in the subendothelial area and in the inner portion of the lamina densa, with gradual tapering of the distribution toward the epithelial side of the basement membrane. The urinary space and epithelial cells were weakly labeled. In tissues from proteinuric patients, albumin was distributed throughout the entire width of the glomerular basement membrane, although the pattern of accumulation varied between patients. The urinary space showed significant labeling associated with some flocculent material. Mesangial areas were heavily labeled in tissues from both control and proteinuric patients. In the latter, lysozomes in glomerular and tubular epithelial cells also accumulated albumin, which is evidence of reabsorption. These results reveal the existence, in normal conditions, of a barrier located in the subendothelial area of the glomerular basement membrane, the loss of which, as in the idiopathic nephrotic syndrome, leads to diffuse distribution of albumin in the glomerular capillary wall. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2260634

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport.

PubMed

Howard, Rebecca J; Carnevale, Vincenzo; Delemotte, Lucie; Hellmich, Ute A; Rothberg, Brad S

2018-04-01

Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process-for example with neuroactive drugs-demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Copyright © 2017 Elsevier B.V. All rights reserved.

NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone

PubMed Central

Awata, Junya; Takada, Saeko; Standley, Clive; Lechtreck, Karl F.; Bellvé, Karl D.; Pazour, Gregory J.; Fogarty, Kevin E.; Witman, George B.

2014-01-01

ABSTRACT The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. An nphp4-null mutant forms flagella with nearly normal length, ultrastructure and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but the amounts of certain membrane proteins were greatly reduced in the mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the transition zone barrier. PMID:25150219

The angiopoietin1-Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8.

PubMed

Liu, Xinchun; Zhou, Xiaoshu; Yuan, Wei

2014-10-15

In mammalian central nervous system (CNS), the integrity of the blood-spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood-brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1-Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8. Copyright © 2014 Elsevier Inc. All rights reserved.

Tunable hydrogen separation in porous graphene membrane: first-principle and molecular dynamic simulation.

PubMed

Tao, Yehan; Xue, Qingzhong; Liu, Zilong; Shan, Meixia; Ling, Cuicui; Wu, Tiantian; Li, Xiaofang

2014-06-11

First-principle density functional theory (DFT) calculation and molecular dynamic (MD) simulation are employed to investigate the hydrogen purification performance of two-dimensional porous graphene material (PG-ESX). First, the pore size of PG-ES1 (3.2775 Å) is expected to show high selectivity of H2 by DFT calculation. Then MD simulations demonstrate the hydrogen purification process of the PG-ESX membrane. The results indicate that the selectivity of H2 over several other gas molecules that often accompany H2 in industrial steam methane reforming or dehydrogenation of alkanes (such as N2, CO, and CH4) is sensitive to the pore size of the membrane. PG-ES and PG-ES1 membranes both exhibit high selectivity for H2 over other gases, but the permeability of the PG-ES membrane is much lower than the PG-ES1 membrane because of the smaller pore size. The PG-ES2 membrane with bigger pores demonstrates low selectivity for H2 over other gases. Energy barrier and electron density have been used to explain the difference of selectivity and permeability of PG-ESX membranes by DFT calculations. The energy barrier for gas molecules passing through the membrane generally increase with the decreasing of pore sizes or increasing of molecule kinetic diameter, due to the different electron overlap between gas and a membrane. The PG-ES1 membrane is far superior to other carbon membranes and has great potential applications in hydrogen purification, energy clean combustion, and making new concept membrane for gas separation.

Potential energy barriers to ion transport within lipid bilayers. Studies with tetraphenylborate.

PubMed Central

Andersen, P S; Fuchs, M

1975-01-01

Tetraphenylborate-induced current transients were studied in lipid bilayers formed from bacterial phosphatidylethanolamine in decane. This ion movement was essentially confined to the membrane in terior during the current transients. Charge movement through the interior of the membrane during the current transients was studied as a function of the applied potential. The transferred charge approached an upper limit with increasing potential, which is interpreted to be the amount of charge due to tetraphenylborate ions absorbed into the boundary regions of the bilayer. A further analysis of the charge transfer as a function of potential indicates that the movement of tetraphenylborate ions is only influenced by a certain farction of the applied potential. For bacterial phosphatidylethanolamine bilayers the effective potential is 77 +/- 4% of the applied potential. The initial conductance and the time constant of the current transients were studied as a function of the applied potential using a Nernst-Planck electrodiffusion regime. It was found that an image-force potential energy barrier gave a good prediction of the observed behavior, provided that the effective potential was used in the calculations. We could not get a satisfactory prediction of the observed behavior with an Eyring rate theory model or a trapezoidal potential energy barrier. PMID:1148364

Transport methods for probing the barrier domain of lipid bilayer membranes.

PubMed Central

Xiang, T X; Chen, X; Anderson, B D

1992-01-01

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent. PMID:1420875

Polyurethane membranes for surgical gown applications

NASA Astrophysics Data System (ADS)

Ukpabi, Pauline Ozoemena

The Occupational Safety and Health Administration (OSHA) recently issued a directive requiring all employers to supply personnel protective equipment to employees who are at risk of exposure to blood or other potentially infectious body fluids. For the healthcare worker, a wide variety of surgical gowns is available commercially but there are concerns over their barrier effectiveness and/or wearer comfort. To successfully create a barrier fabric which combines resistance to fluid penetration with comfort, a complete understanding of the relationship between membrane structure and functional properties is required. In this study, we investigated the surface properties of hydrophilicity and hydrophobicity in polyurethane membranes intended for use in surgical gowns. The polyurethane membranes were grafted with side chains of varying lengths, polyethylene glycol (PEG) being used for the hydrophilic modifications and perfluoroalkyl compounds (a monofunctional acid and a difunctional amino alcohol) for the hydrophobic modifications. The hydrophilic treatment was intended to improve the comfort properties of monolithic membranes without adversely affecting their barrier properties. The hydrophobic treatment, on the other hand, was intended to improve the fluid repellency and hence barrier properties of microporous membranes without adversely affecting their comfort properties. Reflection infrared spectroscopy showed that fluorine was successfully grafted onto the polyurethane backbone during the hydrophobic modification, but was not sensitive enough to detect PEG grafting in leached polyethylene glycol-treated polyurethanes. X-ray photoelectron spectroscopy showed that the perfluoroalkylated polyurethanes contained up to 40% fluorine on their surfaces and the PEG-treated polyurethanes showed an increase in their C-O content over the unmodified polyurethane. Scanning electron microscopy not only showed that perfluoroalkylation yielded polyurethane membranes with very rough surfaces compared to the unmodified membrane, it also showed varying degrees of surface roughness on the perfluoroalkylated polyurethanes depending on whether the monofunctional acid or the difunctional amino alcohol was used as modifier. The PEG-treated samples exhibited smooth surfaces under the SEM. Perfluoroalkylation yielded samples with slightly higher contact angles than the untreated polyurethane while the PEG treatment resulted in polyurethanes with lower contact angles than the untreated polyurethane. The perfluoroalkylated materials were more thermally stable than the unmodified polyurethanes.

In vitro evaluation of electrospun chitosan mats crosslinked with genipin as guided tissue regeneration barrier membranes

NASA Astrophysics Data System (ADS)

Norowski, Peter Andrew, Jr.

Guided tissue regeneration (GTR) is a surgical technique commonly used to exclude bacteria and soft tissues from bone graft sites in oral/maxillofacial bone graft sites by using a barrier membrane to maintain the graft contour and space. Current clinical barrier membrane materials based on expanded polytetrafluoroethylene (ePTFE) and bovine type 1 collagen are non-ideal and experience a number of disadvantages including membrane exposure, bacterial colonization/biofilm formation and premature degradation, all of which result in increased surgical intervention and poor bone regeneration. These materials do not actively participate in tissue regeneration, however bioactive materials, such as chitosan, may provide advantages such as the ability to stimulate wound healing and de novo bone formation. Our hypothesis is that electrospun chitosan GTR membranes will support cell attachment and growth but prevent cell infiltration/penetration of membrane, demonstrate in vitro degradation predictive of 4--6 month in vivo functionality, and will deliver antibiotics locally to prevent/inhibit periopathogenic complications. To test this hypothesis a series of chitosan membranes were electrospun, in the presence or absence of genipin, a natural crosslinking agent, at concentrations of 5 and 10 mM. These membranes were characterized by scanning electron microscopy, tensile testing, suture pullout testing, Fourier transform infrared spectroscopy, X-ray diffraction, and gel permeation chromatography, and in vitro biodegradation for diameter/morphology of fibers, membrane strengths, degree of crosslinking, crystallinity, molecular weight, and degradation kinetics, respectively. Cytocompability of membranes was evaluated in osteoblastic, fibroblastic and monocyte cultures. The activity of minocycline loaded and released from the membranes was determined in zone of inhibition tests using P. gingivalis microbe. The results demonstrated that genipin crosslinking extended the in vitro degradation timeframe, extended the release of minocycline, and increased the tensile strength of the resultant membranes while cytocompatibility, swelling, and tear strength were unaffected. In conclusion, electrospun chitosan membranes crosslinked with genipin are a suitable material for guided tissue regeneration and may help reduce bacterial infection and bacteria-induced host inflammatory response.

Role of lipids in the formation and maintenance of the cutaneous permeability barrier.

PubMed

Feingold, Kenneth R; Elias, Peter M

2014-03-01

The major function of the skin is to form a barrier between the internal milieu and the hostile external environment. A permeability barrier that prevents the loss of water and electrolytes is essential for life on land. The permeability barrier is mediated primarily by lipid enriched lamellar membranes that are localized to the extracellular spaces of the stratum corneum. These lipid enriched membranes have a unique structure and contain approximately 50% ceramides, 25% cholesterol, and 15% free fatty acids with very little phospholipid. Lamellar bodies, which are formed during the differentiation of keratinocytes, play a key role in delivering the lipids from the stratum granulosum cells into the extracellular spaces of the stratum corneum. Lamellar bodies contain predominantly glucosylceramides, phospholipids, and cholesterol and following the exocytosis of lamellar lipids into the extracellular space of the stratum corneum these precursor lipids are converted by beta glucocerebrosidase and phospholipases into the ceramides and fatty acids, which comprise the lamellar membranes. The lipids required for lamellar body formation are derived from de novo synthesis by keratinocytes and from extra-cutaneous sources. The lipid synthetic pathways and the regulation of these pathways are described in this review. In addition, the pathways for the uptake of extra-cutaneous lipids into keratinocytes are discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias. Published by Elsevier B.V.

Exploring the role of peptides in polymer-based gene delivery.

PubMed

Sun, Yanping; Yang, Zhen; Wang, Chunxi; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian

2017-09-15

Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors. Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition‐related alterations

PubMed Central

Rühl, Jana; Hein, Eva‐Maria; Hayen, Heiko; Schmid, Andreas; Blank, Lars M.

2012-01-01

Summary Microorganisms, such as Pseudomonas putida, utilize specific physical properties of cellular membrane constituents, mainly glycerophospholipids, to (re‐)adjust the membrane barrier to environmental stresses. Building a basis for membrane composition/function studies, we inventoried the glycerophospholipids of different Pseudomonas and challenged membranes of growing cells with n‐butanol. Using a new high‐resolution liquid chromatography/mass spectrometry (LC/MS) method, 127 glycerophospholipid species [e.g. phosphatidylethanolamine PE(32:1)] with up to five fatty acid combinations were detected. The glycerophospholipid inventory consists of 305 distinct glycerophospholipids [e.g. PE(16:0/16:1)], thereof 14 lyso‐glycerophospholipids, revealing conserved compositions within the four investigated pseudomonads P. putida KT2440, DOT‐T1E, S12 and Pseudomonas sp. strain VLB120. Furthermore, we addressed the influence of environmental conditions on the glycerophospholipid composition of Pseudomonas via long‐time exposure to the sublethal n‐butanol concentration of 1% (v/v), focusing on: (i) relative amounts of glycerophospholipid species, (ii) glycerophospholipid head group composition, (iii) fatty acid chain length, (iv) degree of saturation and (v) cis/trans isomerization of unsaturated fatty acids. Observed alterations consist of changing head group compositions and for the solvent‐sensitive strain KT2440 diminished fatty acid saturation degrees. Minor changes in the glycerophospholipid composition of the solvent‐tolerant strains P. putida S12 and Pseudomonas sp. VLB120 suggest different strategies of the investigated Pseudomonas to maintain the barrier function of cellular membranes. PMID:21895997

Novel sulfonated polyimide/zwitterionic polymer-functionalized graphene oxide hybrid membranes for vanadium redox flow battery

NASA Astrophysics Data System (ADS)

Cao, Li; Kong, Lei; Kong, Lingqian; Zhang, Xingxiang; Shi, Haifeng

2015-12-01

Hybrid membranes (SPI/ZGO) composed of sulfonated polyimide (SPI) and zwitterionic polymer-functionalized graphene oxide (ZGO) are fabricated via a solution-casting method for vanadium redox flow battery (VRB). Successful preparation of ZGO fillers and SPI/ZGO hybrid membranes are demonstrated by FT-IR, XPS and SEM, indicating that ZGO fillers is homogeneously dispersed into SPI matrix. Through controlling the interfacial interaction between SPI matrix and ZGO fillers, the physicochemical properties, e.g., vanadium ion barrier and proton transport pathway, of hybrid membranes are tuned via the zwitterionic acid-base interaction in the hybrid membrane, showing a high ion selectivity and good stability with the incorporated ZGO fillers. SPI/ZGO-4 hybrid membrane proves a higher cell efficiencies (CE: 92-98%, EE: 65-79%) than commercial Nafion 117 membrane (CE: 89-94%, EE: 59-70%) for VRB application at 30-80 mA cm-2. The assembled VRB with SPI/ZGO-4 membrane presents a stable cycling charge-discharge performance over 280 times, which demonstrates its excellent chemical stability under the strong acidic and oxidizing conditions. SPI/ZGO hybrid membranes show a brilliant perspective for VRB application.

Molecular dynamics studies of simple membrane-water interfaces: Structure and functions in the beginnings of cellular life

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Wilson, Michael A.

1995-01-01

Molecular dynamics computer simulations of the structure and functions of a simple membrane are performed in order to examine whether membranes provide an environment capable of promoting protobiological evolution. Our model membrane is composed of glycerol 1-monooleate. It is found that the bilayer surface fluctuates in time and space, occasionally creating thinning defects in the membrane. These defects are essential for passive transport of simple ions across membranes because they reduce the Born barrier to this process by approximately 40%. Negative ions are transferred across the bilayer more readily than positive ions due to favorable interactions with the electric field at the membrane-water interface. Passive transport of neutral molecules is, in general, more complex than predicted by the solubility-diffusion model. In particular, molecules which exhibit sufficient hydrophilicity and lipophilicity concentrate near membrane surfaces and experience 'interfacial resistance' to transport. The membrane-water interface forms an environment suitable for heterogeneous catalysis. Several possible mechanisms leading to an increase of reaction rates at the interface are discussed. We conclude that vesicles have many properties that make them very good candidates for earliest protocells. Some potentially fruitful directions of experimental and theoretical research on this subject are proposed.

Tight junctions and the modulation of barrier function in disease

PubMed Central

2008-01-01

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease. PMID:18415116

Supported Lipid Bilayer/Carbon Nanotube Hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose; Craighead, Harold; McEuen, Paul

2007-03-01

We form supported lipid bilayers on single-walled carbon nanotubes and use this hybrid structure to probe the properties of lipid membranes and their functional constituents. We first demonstrate membrane continuity and lipid diffusion over the nanotube. A membrane-bound tetanus toxin protein, on the other hand, sees the nanotube as a diffusion barrier whose strength depends on the diameter of the nanotube. Finally, we present results on the electrical detection of specific binding of streptavidin to biotinylated lipids with nanotube field effect transistors. Possible techniques to extract dynamic information about the protein binding events will also be discussed.

How hormones influence composition and physiological function of the brain-blood barrier.

PubMed

Hampl, R; Bičíková, M; Sosvorová, L

2015-01-01

Hormones exert many actions in the brain. Their access and effects in the brain are regulated by the blood-brain barrier (BBB). Hormones as other substances may enter the brain and vice versa either by paracellular way requiring breaching tight junctions stitching the endothelial cells composing the BBB, or by passage through the cells (transcellular way). Hormones influence both ways through their receptors, both membrane and intracellular, present on/in the BBB. In the review the main examples are outlined how hormones influence the expression and function of proteins forming the tight junctions, as well as how they regulate expression and function of major protein transporters mediating transport of various substances including hormone themselves.

Efflux proteins at the blood-brain barrier: review and bioinformatics analysis.

PubMed

Saidijam, Massoud; Karimi Dermani, Fatemeh; Sohrabi, Sareh; Patching, Simon G

2018-05-01

1. Efflux proteins at the blood-brain barrier provide a mechanism for export of waste products of normal metabolism from the brain and help to maintain brain homeostasis. They also prevent entry into the brain of a wide range of potentially harmful compounds such as drugs and xenobiotics. 2. Conversely, efflux proteins also hinder delivery of therapeutic drugs to the brain and central nervous system used to treat brain tumours and neurological disorders. For bypassing efflux proteins, a comprehensive understanding of their structures, functions and molecular mechanisms is necessary, along with new strategies and technologies for delivery of drugs across the blood-brain barrier. 3. We review efflux proteins at the blood-brain barrier, classified as either ATP-binding cassette (ABC) transporters (P-gp, BCRP, MRPs) or solute carrier (SLC) transporters (OATP1A2, OATP1A4, OATP1C1, OATP2B1, OAT3, EAATs, PMAT/hENT4 and MATE1). 4. This includes information about substrate and inhibitor specificity, structural organisation and mechanism, membrane localisation, regulation of expression and activity, effects of diseases and conditions and the principal technique used for in vivo analysis of efflux protein activity: positron emission tomography (PET). 5. We also performed analyses of evolutionary relationships, membrane topologies and amino acid compositions of the proteins, and linked these to structure and function.

A simple cell transport device keeps culture alive and functional during shipping.

PubMed

Miller, Paula G; Wang, Ying I; Swan, Glen; Shuler, Michael L

2017-09-01

Transporting living complex cellular constructs through the mail while retaining their full viability and functionality is challenging. During this process, cells often suffer from exposure to suboptimal life-sustaining conditions (e.g. temperature, pH), as well as damage due to shear stress. We have developed a transport device for shipping intact cell/tissue constructs from one facility to another that overcomes these obstacles. Our transport device maintained three different cell lines (Caco2, A549, and HepG2 C3A) individually on transwell membranes with high viability (above 97%) for 48 h under simulated shipping conditions without an incubator. The device was also tested by actual overnight shipping of blood brain barrier constructs consisting of human induced pluripotent brain microvascular endothelial cells and rat astrocytes on transwell membranes to a remote facility (approximately 1200 miles away). The blood brain barrier constructs arrived with high cell viability and were able to regain full barrier integrity after equilibrating in the incubator for 24 h; this was assessed by the presence of continuous tight junction networks and in vivo-like values for trans-endothelial electrical resistance (TEER). These results demonstrated that our cell transport device could be a useful tool for long-distance transport of membrane-bound cell cultures and functional tissue constructs. Studies that involve various cell and tissue constructs, such as the "Multi-Organ-on-Chip" devices (where multiple microscale tissue constructs are integrated on a single microfluidic device) and studies that involve microenvironments where multiple tissue interactions are of interest, would benefit from the ability to transport or receive these constructs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1257-1266, 2017. © 2017 American Institute of Chemical Engineers.

Delineation of Matriptase Protein Expression by Enzymatic Gene Trapping Suggests Diverging Roles in Barrier Function, Hair Formation, and Squamous Cell Carcinogenesis

PubMed Central

List, Karin; Szabo, Roman; Molinolo, Alfredo; Nielsen, Boye Schnack; Bugge, Thomas H.

2006-01-01

The membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma. PMID:16651618

Delineation of matriptase protein expression by enzymatic gene trapping suggests diverging roles in barrier function, hair formation, and squamous cell carcinogenesis.

PubMed

List, Karin; Szabo, Roman; Molinolo, Alfredo; Nielsen, Boye Schnack; Bugge, Thomas H

2006-05-01

The membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma.

Evaluation of novel resorbable membranes for bone augmentation in a rat model.

PubMed

Zeng, Ni; van Leeuwen, Anne; Yuan, Huipin; Bos, Ruud R M; Grijpma, Dirk W; Kuijer, Roel

2016-02-01

Our study compared two novel, biodegradable poly(trimethylene carbonate) (PTMC) barrier membranes to clinically applied barrier membranes in maintaining volume of block autologous bone grafts in a rat mandible model. Two hundred and forty rats were included in this study. Block autologous bone grafts of 5 mm in diameter were harvested from the mandibular angles and transplanted onto the contralateral side. The bone grafts were either covered with a membrane or left uncovered. The applied membranes included pure PTMC membranes, biphasic calcium phosphate (BCP) incorporated PTMC composite membranes, expanded poly(tetrafluoroethylene) (e-PTFE) membranes (Tex) and collagen membranes (Geistlich Bio-Gide). After 2, 4 and 12 weeks, the rat mandibles were retrieved and analysed by histological evaluation and μCT quantification. The histological evaluation revealed that in time the block autologous bone graft was well integrated to the recipient bone via gradually maturing newly formed bone and did not show signs of resorption, independent of membrane coverage or types of membrane. μCT quantification showed the volume of the bone graft and recipient bone together was maintained by new bone formation and recipient bone resorption. Our study showed that the use of PTMC membranes and PTMC-BCP composite membranes resulted in similar bone remodelling to the collagen membranes and e-PTFE membranes and that the use of barrier membranes did not interfere with bone remodelling of the bone grafts and recipient bones. However, the used barrier membranes seemed not to contribute in maintaining the volume of block autologous bone grafts. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Acrosome reaction inducers impose alterations in repulsive strain and hydration barrier in human sperm membranes.

PubMed

Purohit, S B; Laloraya, M; Kumar, G P

1998-06-01

Spin labeling studies of the lipophilic domains of human spermatozoa during capacitation and during acrosome reaction (AR) under the influence of selected AR-inducers were performed. Significantly enhanced rotational function of molecules was obvious during capacitation, with no significant changes in membrane packaging or the lateral diffusion of molecules. The AR inducers appeared to restrict the rotational freedom of molecules, dramatically enhancing the lateral diffusion and ordering coefficients. A significant decrease in superoxide anion generation was observed in the acrosome reacted groups when compared to the non-acrosome reacted groups. A high level of superoxide anion radical (O2.-) level maintained in capacitated spermatozoa would add to the Van der Waal's repulsive forces at the polar head of phospholipids, holding the membrane in strain where the molecular enjoy little freedom for lateral motion. A sudden drop in the levels of O2.- in spermatozoa upon addition of AR inducers could abruptly release the local hydrophobic repulsive strain within the membrane. This loss of hydration barrier explains the observed enhancement in lateral diffusion profiles of lipids and the packaging of molecules. It is reasonable to assume that these phenomena could be amplified further by interplay of Ca2+ by modifying the local charge aggregation. Thus, we would conclude that AR inducers release the oxyradical load in capacitated spermatozoa, which would modify the repulsive strain and hydration barrier forces in the lipophilic domains permitting vesiculation of the membranes. It appears that various acrosome reaction inducers act as effectors of grossly similar physical alterations in sperm membranes and that the resulting signal cascades proceed through intercalating biochemical sequences.

Interaction of Local Anesthetics with Biomembranes Consisting of Phospholipids and Cholesterol: Mechanistic and Clinical Implications for Anesthetic and Cardiotoxic Effects

PubMed Central

2013-01-01

Despite a long history in medical and dental application, the molecular mechanism and precise site of action are still arguable for local anesthetics. Their effects are considered to be induced by acting on functional proteins, on membrane lipids, or on both. Local anesthetics primarily interact with sodium channels embedded in cell membranes to reduce the excitability of nerve cells and cardiomyocytes or produce a malfunction of the cardiovascular system. However, the membrane protein-interacting theory cannot explain all of the pharmacological and toxicological features of local anesthetics. The administered drug molecules must diffuse through the lipid barriers of nerve sheaths and penetrate into or across the lipid bilayers of cell membranes to reach the acting site on transmembrane proteins. Amphiphilic local anesthetics interact hydrophobically and electrostatically with lipid bilayers and modify their physicochemical property, with the direct inhibition of membrane functions, and with the resultant alteration of the membrane lipid environments surrounding transmembrane proteins and the subsequent protein conformational change, leading to the inhibition of channel functions. We review recent studies on the interaction of local anesthetics with biomembranes consisting of phospholipids and cholesterol. Understanding the membrane interactivity of local anesthetics would provide novel insights into their anesthetic and cardiotoxic effects. PMID:24174934

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

PubMed

Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

2015-01-01

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

PubMed

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. How ATP hydrolysis is coupled with LPS release from the membrane is not understood. We have identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport, some of which coordinate ATPase activity with LPS release. Copyright © 2016 Simpson et al.

Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

NASA Astrophysics Data System (ADS)

Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

2017-12-01

In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5-3 µm were used and a shear stress of ~0.03 dyne cm-2 was created by applying a low flow rate of 20 nl s-1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

Adjunctive Effect of Autologus Platelet-Rich Fibrin to Barrier Membrane in the Treatment of Periodontal Intrabony Defects.

PubMed

Panda, Saurav; Sankari, Malaiappan; Satpathy, Anurag; Jayakumar, Doraiswamy; Mozzati, Marco; Mortellaro, Carmen; Gallesio, Giorgia; Taschieri, Silvio; Del Fabbro, Massimo

2016-05-01

Autologous platelet-rich fibrin (PRF) and barrier membranes in the treatment of intrabony defects in chronic periodontitis patients have shown significant clinical benefits. This study evaluates the additive effect of autologous PRF in combination with a barrier membrane versus the use of barrier membrane alone for the treatment of intrabony defects in chronic periodontitis patients. A randomized split-mouth design was used. Sixteen patients with 32 paired intrabony defects were included. In each patient 1 defect was treated using a resorbable collagen membrane along with PRF (test group) and the other defect by guided tissue regeneration alone (control group). The following clinical parameters were measured at baseline and after 9 months: plaque index, modified sulcus bleeding index, probing pocket depth, clinical attachment level, and gingival marginal level. The radiographic defect depth was also assessed at baseline and after 9 months. Test group showed a statistically significant improvement for probing depth (P = 0.002), clinical attachment level (P = 0.001), and radiographic defect depth (P < 0.001) after 9 months as compared with the control sites. Radiographic defect depth reduction was 58.19 ± 13.24% in the test group as compared with 24.86 ± 9.94% reduction in the control group. The adjunctive use of PRF in combination with barrier membrane is more effective in the treatment of intrabony defects in chronic periodontitis as compared with barrier membrane alone.

Mapping the membrane-aqueous border for the voltage-sensing domain of a potassium channel.

PubMed

Neale, Edward J; Rong, Honglin; Cockcroft, Christopher J; Sivaprasadarao, Asipu

2007-12-28

Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (~28 A), but those of S3 and S4 are considerably shorter (> or = 40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.

Interactions of a Charged Nanoparticle with a Lipid Membrane: Implications for Gene Delivery

PubMed Central

Ting, Christina L.; Wang, Zhen-Gang

2011-01-01

We employ self-consistent field theory to study the thermodynamics of membrane-particle interactions in the context of gene delivery systems, with the aim to guide the design of dendrimers that can overcome the endosomal escape barrier by inserting into membranes and creating pores. We consider the interaction between a model polyamidoamine dendrimer and a membrane under controlled tension as a function of the separation between the dendrimer and the membrane. In all the cases we have studied, the lowest free energy state corresponds to the membrane partially wrapping the dendrimer. However, with moderate tension, we find that a G5 (or larger) generation dendrimer, through thermal fluctuation, can induce the formation of metastable pores. These metastable pores are subsequently shown to significantly lower the critical tension necessary for membrane rupture, thus possibly enhancing the release of the trapped genetic material from the endosome. PMID:21354402

A Key Claudin Extracellular Loop Domain is Critical for Epithelial Barrier Integrity

PubMed Central

Mrsny, Randall J.; Brown, G. Thomas; Gerner-Smidt, Kirsten; Buret, Andre G.; Meddings, Jon B.; Quan, Clifford; Koval, Michael; Nusrat, Asma

2008-01-01

Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-153–80) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-153–80 associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-153–80, because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-153–80 to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-153–80, capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier. PMID:18349130

The relevance of polymeric synthetic membranes in topical formulation assessment and drug diffusion study.

PubMed

Ng, Shiow-Fern; Rouse, Jennifer J; Sanderson, Francis D; Eccleston, Gillian M

2012-03-01

Synthetic membranes are composed of thin sheets of polymeric macromolecules that can control the passage of components through them. Generally, synthetic membranes used in drug diffusion studies have one of two functions: skin simulation or quality control. Synthetic membranes for skin simulation, such as the silicone-based membranes polydimethylsiloxane and Carbosil, are generally hydrophobic and rate limiting, imitating the stratum corneum. In contrast, synthetic membranes for quality control, such as cellulose esters and polysulfone, are required to act as a support rather than a barrier. These synthetic membranes also often contain pores; hence, they are called porous membranes. The significance of Franz diffusion studies and synthetic membranes in quality control studies involves an understanding of the fundamentals of synthetic membranes. This article provides a general overview of synthetic membranes, including a brief background of the history and the common applications of synthetic membranes. This review then explores the types of synthetic membranes, the transport mechanisms across them, and their relevance in choosing a synthetic membrane in Franz diffusion cell studies for formulation assessment purposes.

Membrane fouling in a submerged membrane bioreactor: effect of pH and its implications.

PubMed

Zhang, Ye; Zhang, Meijia; Wang, Fangyuan; Hong, Huachang; Wang, Aijun; Wang, Juan; Weng, Xuexiang; Lin, Hongjun

2014-01-01

The effect of pH on membrane fouling in a submerged membrane bioreactor (MBR) was investigated in this study. It was found that, pH increase slightly increased the resistance of virgin membrane and fouled membrane. Pore clogging resistance was quite low, which was not apparently affected by the pH variation. Lower pH resulted in higher adherence of sludge flocs on membrane surface. Thermodynamic analysis showed that a repulsive energy barrier existed in the process of the foulants approaching to membrane surface. This energy barrier would decrease with pH decreased, suggesting the existence of a critical pH below which the repulsive energy barrier would disappear, which would facilitate attachment of the foulants. The resistance of the formed cake layer would significantly increase with the feed pH. This result could be explained by the osmotic pressure mechanism. The obtained findings also provided important implications for membrane fouling mitigation in MBRs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

PubMed Central

Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

2015-01-01

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

Position-Dependent Diffusion Tensors in Anisotropic Media from Simulation: Oxygen Transport in and through Membranes.

PubMed

Ghysels, An; Venable, Richard M; Pastor, Richard W; Hummer, Gerhard

2017-06-13

A Bayesian-based methodology is developed to estimate diffusion tensors from molecular dynamics simulations of permeants in anisotropic media, and is applied to oxygen in lipid bilayers. By a separation of variables in the Smoluchowski diffusion equation, the multidimensional diffusion is reduced to coupled one-dimensional diffusion problems that are treated by discretization. The resulting diffusivity profiles characterize the membrane transport dynamics as a function of the position across the membrane, discriminating between diffusion normal and parallel to the membrane. The methodology is first validated with neat water, neat hexadecane, and a hexadecane slab surrounded by water, the latter being a simple model for a lipid membrane. Next, a bilayer consisting of pure 1-palmitoyl 2-oleoylphosphatidylcholine (POPC), and a bilayer mimicking the lipid composition of the inner mitochondrial membrane, including cardiolipin, are investigated. We analyze the detailed time evolution of oxygen molecules, in terms of both normal diffusion through and radial diffusion inside the membrane. Diffusion is fast in the more loosely packed interleaflet region, and anisotropic, with oxygen spreading more rapidly in the membrane plane than normal to it. Visualization of the propagator shows that oxygen enters the membrane rapidly, reaching its thermodynamically favored center in about 1 ns, despite the free energy barrier at the headgroup region. Oxygen transport is quantified by computing the oxygen permeability of the membranes and the average radial diffusivity, which confirm the anisotropy of the diffusion. The position-dependent diffusion constants and free energies are used to construct compartmental models and test assumptions used in estimating permeability, including Overton's rule. In particular, a hexadecane slab surrounded by water is found to be a poor model of oxygen transport in membranes because the relevant energy barriers differ substantially.

Molecular dynamics simulation of the evolution of hydrophobic defects in one monolayer of a phosphatidylcholine bilayer: relevance for membrane fusion mechanisms.

PubMed Central

Tieleman, D Peter; Bentz, Joe

2002-01-01

The spontaneous formation of the phospholipid bilayer underlies the permeability barrier function of the biological membrane. Tears or defects that expose water to the acyl chains are spontaneously healed by lipid lateral diffusion. However, mechanical barriers, e.g., protein aggregates held in place, could sustain hydrophobic defects. Such defects have been postulated to occur in processes such as membrane fusion. This gives rise to a new question in bilayer structure: What do the lipids do in the absence of lipid lateral diffusion to minimize the free energy of a hydrophobic defect? As a first step to understand this rather fundamental question about bilayer structure, we performed molecular dynamic simulations of up to 10 ns of a planar bilayer from which lipids have been deleted randomly from one monolayer. In one set of simulations, approximately one-half of the lipids in the defect monolayer were restrained to form a mechanical barrier. In the second set, lipids were free to diffuse around. The question was simply whether the defects caused by removing a lipid would aggregate together, forming a large hydrophobic cavity, or whether the membrane would adjust in another way. When there are no mechanical barriers, the lipids in the defect monolayer simply spread out and thin with little effect on the other intact monolayer. In the presence of a mechanical barrier, the behavior of the lipids depends on the size of the defect. When 3 of 64 lipids are removed, the remaining lipids adjust the lower one-half of their chains, but the headgroup structure changes little and the intact monolayer is unaffected. When 6 to 12 lipids are removed, the defect monolayer thins, lipid disorder increases, and lipids from the intact monolayer move toward the defect monolayer. Whereas this is a highly simplified model of a fusion site, this engagement of the intact monolayer into the fusion defect is strikingly consistent with recent results for influenza hemagglutinin mediated fusion. PMID:12202375

Challenges in Commercializing Biomimetic Membranes

PubMed Central

Perry, Mark; Madsen, Steen Ulrik; Jørgensen, Tine; Braekevelt, Sylvie; Lauritzen, Karsten; Hélix-Nielsen, Claus

2015-01-01

The discovery of selective water channel proteins—aquaporins—has prompted growing interest in using these proteins, as the building blocks for designing new types of membranes. However, as with any other new and potentially disruptive technology, barriers for successful market entry exist. One category includes customer-related barriers, which can be influenced to some extent. Another category includes market-technical-related barriers, which can be very difficult to overcome by an organization/company aiming at successfully introducing their innovation on the market—in particular if both the organization and the technology are at early stages. Often, one faces barriers from both these categories at the same time, which makes it necessary to gain insight of the particular market when introducing a new innovative product. In this review we present the basic concepts and discuss some of these barriers and challenges associated with introducing biomimetic aquaporin membranes. These include technical issues in membrane production and product testing. Then we discuss possible business models for introducing new technologies in general, followed by a presentation of beach-head market segments relevant for biomimetic aquaporin membranes. PMID:26556379

Challenges in Commercializing Biomimetic Membranes.

PubMed

Perry, Mark; Madsen, Steen Ulrik; Jørgensen, Tine; Braekevelt, Sylvie; Lauritzen, Karsten; Hélix-Nielsen, Claus

2015-11-05

The discovery of selective water channel proteins-aquaporins-has prompted growing interest in using these proteins, as the building blocks for designing new types of membranes. However, as with any other new and potentially disruptive technology, barriers for successful market entry exist. One category includes customer-related barriers, which can be influenced to some extent. Another category includes market-technical-related barriers, which can be very difficult to overcome by an organization/company aiming at successfully introducing their innovation on the market-in particular if both the organization and the technology are at early stages. Often, one faces barriers from both these categories at the same time, which makes it necessary to gain insight of the particular market when introducing a new innovative product. In this review we present the basic concepts and discuss some of these barriers and challenges associated with introducing biomimetic aquaporin membranes. These include technical issues in membrane production and product testing. Then we discuss possible business models for introducing new technologies in general, followed by a presentation of beach-head market segments relevant for biomimetic aquaporin membranes.

Acyl chain asymmetry and polyunsaturation of brain phospholipids facilitate membrane vesiculation without leakage

PubMed Central

Manni, Marco M; Tiberti, Marion L; Pagnotta, Sophie; Barelli, Hélène; Gautier, Romain

2018-01-01

Phospholipid membranes form cellular barriers but need to be flexible enough to divide by fission. Phospholipids generally contain a saturated fatty acid (FA) at position sn1 whereas the sn2-FA is saturated, monounsaturated or polyunsaturated. Our understanding of the impact of phospholipid unsaturation on membrane flexibility and fission is fragmentary. Here, we provide a comprehensive view of the effects of the FA profile of phospholipids on membrane vesiculation by dynamin and endophilin. Coupled to simulations, this analysis indicates that: (i) phospholipids with two polyunsaturated FAs make membranes prone to vesiculation but highly permeable; (ii) asymmetric sn1-saturated-sn2-polyunsaturated phospholipids provide a tradeoff between efficient membrane vesiculation and low membrane permeability; (iii) When incorporated into phospholipids, docosahexaenoic acid (DHA; omega-3) makes membranes more deformable than arachidonic acid (omega-6). These results suggest an explanation for the abundance of sn1-saturated-sn2-DHA phospholipids in synaptic membranes and for the importance of the omega-6/omega-3 ratio on neuronal functions. PMID:29543154

Microvillar Ca++ signaling: a new view of an old problem.

PubMed

Lange, K

1999-07-01

Proceeding from the recent finding that the main components of the Ca++ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca++ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca++ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca++ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca++ signaling. It combines the function of a diffusion barrier, preventing Ca++ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP3-sensitive Ca++ store. Activation of Ca++ signaling via PLC-coupled receptors simultaneously empties Ca++ stores and activates the influx of external Ca++. The presented concept of Ca++ signaling is compatible with all established data on Ca++ signaling. Properties of Ca++ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca++ release, Ca(++)-induced Ca++ release (CICR), the coupling phenomen between the filling state of the Ca++ store and the activity of the Ca++ influx pathway, as well as the various yet unexplained complex kinetics of Ca++ uptake and release can be explained on a common mechanistic basis.

Alterations in blood-brain barrier function following acute hypertension: comparison of the blood-to-brain transfer of horseradish peroxidase with that of alpha-aminisobutyric acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ellison, M.D.B.

The blood-brain barrier (BBB) selectively restricts the blood-to-brain passage of many solutes owing to unique properties of cerebrovascular endothelial cell membranes. To date, experimental study of the BBB has been accomplished primarily through the use of two different methodological approaches. Morphological studies have mostly employed large molecular weight (MW) tracers to detect morphological alterations underlying increased permeability. Physiological studies, employing smaller, more physiologic tracers have successfully described, quantitatively, certain functional aspects of blood-to-brain transfer. The current work attempts to merge these two approaches and to consider barrier function/dysfunction from both a morphological and a functional perspective. Specifically, the study comparesmore » in rats, following acute hypertension, the cerebrovascular passage of /sup 14/C-alpha-aminoisobutyric acid (AIB) and that of horseradish peroxidase (HRP). The blood-to-brain passage of AIB and HRP were compared following acute hypertension, with regard to both the distributions of the tracer extravasation patterns and the magnitude of tracer extravasation. The results of this study suggest that traditional morphological barrier studies alone do not reveal all aspects of altered barrier status and that multiple mechanisms underlying increased BBB permeability may operate simultaneously during BBB dysfunction.« less

Hydroxide Degradation Pathways for Substituted Benzyltrimethyl Ammonium: A DFT Study

DOE PAGES

Long, Hai; Pivovar, Bryan S.

2014-11-01

The stability of cations used in the alkaline exchange membranes has been a major challenge. In this paper, degradation energy barriers were investigated by density functional theory for substituted benzyltrimethyl ammonium (BTMA+) cations. Findings show that electron-donating substituent groups at meta-position(s) of the benzyl ring could result in increased degradation barriers. However, after investigating more than thirty substituted BTMA+ cations, the largest improvement in degradation barrier found was only 6.7 kJ/mol. This suggests a modest (8×) improvement in stability for this type of approach may be possible, but for anything greater other approaches will need to be pursued.

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

PubMed Central

Zhang, Xi

2015-01-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

Potential Applications of Phyto-Derived Ceramides in Improving Epidermal Barrier Function.

PubMed

Tessema, Efrem N; Gebre-Mariam, Tsige; Neubert, Reinhard H H; Wohlrab, Johannes

2017-01-01

The outer most layer of the skin, the stratum corneum, consists of corneocytes which are coated by a cornified envelope and embedded in a lipid matrix of ordered lamellar structure. It is responsible for the skin barrier function. Ceramides (CERs) are the backbone of the intercellular lipid membranes. Skin diseases such as atopic dermatitis and psoriasis and aged skin are characterized by dysfunctional skin barrier and dryness which are associated with reduced levels of CERs. Previously, the effectiveness of supplementation of synthetic and animal-based CERs in replenishing the depleted natural skin CERs and restoring the skin barrier function have been investigated. Recently, however, the barrier function improving effect of plant-derived CERs has attracted much attention. Phyto-derived CERs (phytoCERs) are preferable due to their assumed higher safety as they are mostly isolated from dietary sources. The beneficial effects of phytoCER-based oral dietary supplements for skin hydration and skin barrier reinforcement have been indicated in several studies involving animal models as well as human subjects. Ingestible dietary supplements containing phytoCERs are also widely available on the market. Nonetheless, little effort has been made to investigate the potential cosmetic applications of topically administered phytoCERs. Therefore, summarizing the foregoing investigations and identifying the gap in the scientific data on plant-derived CERs intended for skin-health benefits are of paramount importance. In this review, an attempt is made to synthesize the information available in the literature regarding the effects of phytoCER-based oral dietary supplements on skin hydration and barrier function with the underlying mechanisms. © 2017 S. Karger AG, Basel.

Claudins and the Modulation of Tight Junction Permeability

PubMed Central

Günzel, Dorothee

2013-01-01

Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions. PMID:23589827

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Strategies in biomimetic surface engineering of nanoparticles for biomedical applications

NASA Astrophysics Data System (ADS)

Gong, Yong-Kuan; Winnik, Françoise M.

2012-01-01

Engineered nanoparticles (NPs) play an increasingly important role in biomedical sciences and in nanomedicine. Yet, in spite of significant advances, it remains difficult to construct drug-loaded NPs with precisely defined therapeutic effects, in terms of release time and spatial targeting. The body is a highly complex system that imposes multiple physiological and cellular barriers to foreign objects. Upon injection in the blood stream or following oral administation, NPs have to bypass numerous barriers prior to reaching their intended target. A particularly successful design strategy consists in masking the NP to the biological environment by covering it with an outer surface mimicking the composition and functionality of the cell's external membrane. This review describes this biomimetic approach. First, we outline key features of the composition and function of the cell membrane. Then, we present recent developments in the fabrication of molecules that mimic biomolecules present on the cell membrane, such as proteins, peptides, and carbohydrates. We present effective strategies to link such bioactive molecules to the NPs surface and we highlight the power of this approach by presenting some exciting examples of biomimetically engineered NPs useful for multimodal diagnostics and for target-specific drug/gene delivery applications. Finally, critical directions for future research and applications of biomimetic NPs are suggested to the readers.

Viral membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

Effect of acidic aqueous solution on chemical and physical properties of polyamide NF membranes

NASA Astrophysics Data System (ADS)

Jun, Byung-Moon; Kim, Su Hwan; Kwak, Sang Kyu; Kwon, Young-Nam

2018-06-01

This work was systematically investigated the effects of acidic aqueous solution (15 wt% sulfuric acid as model wastewater from smelting process) on the physical and chemical properties of commercially available nanofiltration (NF) polyamide membranes, using piperazine (PIP)-based NE40/70 membranes and m-phenylene diamine (MPD)-based NE90 membrane. Surface properties of the membranes were studied before and after exposure to strong acid using various analytical tools: Scanning Electron Microscopy (SEM), Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), contact angle analyzer, and electrophoretic light scattering spectrophotometer. The characterization and permeation results showed piperazine-based NE40/70 membranes have relatively lower acid-resistance than MPD-based NE90 membrane. Furthermore, density functional theory (DFT) calculation was also conducted to reveal the different acid-tolerances between the piperazine-based and MPD-based polyamide membranes. The easiest protonation was found to be the protonation of oxygen in piperazine-based monomer, and the N-protonation of the monomer had the lowest energy barrier in the rate determining step (RDS). The calculations were well compatible with the surface characterization results. In addition, the energy barrier in RDS is highly correlated with the twist angle (τD), which determines the delocalization of electrons between the carbonyl πCO bond and nitrogen lone pair, and the tendency of the twist angle was also maintained in longer molecules (dimer and trimer). This study clearly explained why the semi-aromatic membrane (NE40/70) is chemically less stable than the aromatic membrane (NE90) given the surface characterizations and DFT calculation results.

Age-dependent changes at the blood-brain barrier. A Comparative structural and functional study in young adult and middle aged rats.

PubMed

Bors, Luca; Tóth, Kinga; Tóth, Estilla Zsófia; Bajza, Ágnes; Csorba, Attila; Szigeti, Krisztián; Máthé, Domokos; Perlaki, Gábor; Orsi, Gergely; Tóth, Gábor K; Erdő, Franciska

2018-05-01

Decreased beta-amyloid clearance in Alzheimer's disease and increased blood-brain barrier permeability in aged subjects have been reported in several articles. However, morphological and functional characterization of blood-brain barrier and its membrane transporter activity have not been described in physiological aging yet. The aim of our study was to explore the structural changes in the brain microvessels and possible functional alterations of P-glycoprotein at the blood-brain barrier with aging. Our approach included MR imaging for anatomical orientation in middle aged rats, electronmicroscopy and immunohistochemistry to analyse the alterations at cellular level, dual or triple-probe microdialysis and SPECT to test P-glycoprotein functionality in young and middle aged rats. Our results indicate that the thickness of basal lamina increases, the number of tight junctions decreases and the size of astrocyte endfeet extends with advanced age. On the basis of microdialysis and SPECT results the P-gp function is reduced in old rats. With our multiparametric approach a complex regulation can be suggested which includes elements leading to increased permeability of blood-brain barrier by enhanced paracellular and transcellular transport, and factors working against it. To verify the role of P-gp pumps in brain aging further studies are warranted. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

S-nitrosylation of VASP at cysteine 64 mediates the inflammation-stimulated increase in microvascular permeability.

PubMed

Zamorano, Patricia; Marín, Natalie; Córdova, Francisco; Aguilar, Alejandra; Meininger, Cynthia; Boric, Mauricio P; Golenhofen, Nikola; Contreras, Jorge E; Sarmiento, José; Durán, Walter N; Sánchez, Fabiola A

2017-07-01

We tested the hypothesis that platelet-activating factor (PAF) induces S -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated S -nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S -nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that S -nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. S -nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist. NEW & NOTEWORTHY Here, we demonstrate that S -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that S -nitrosylation of VASP contributes to the onset of endothelial permeability. Copyright © 2017 the American Physiological Society.

Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction

PubMed Central

Terriza, Antonia; Vilches-Pérez, José I.; González-Caballero, Juan L.; de la Orden, Emilio; Yubero, Francisco; Barranco, Angel; Gonzalez-Elipe, Agustín R.; Vilches, José; Salido, Mercedes

2014-01-01

New biomaterials for Guided Bone Regeneration (GBR), both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB®) HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide) (PLGA) membrane foil functionalized by a very thin film (around 15 nm) of TiO2 (i.e., TiO2/PLGA membranes), designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors. PMID:28788538

Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

PubMed Central

Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

2014-01-01

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

Bone conditioned media (BCM) improves osteoblast adhesion and differentiation on collagen barrier membranes.

PubMed

Fujioka-Kobayashi, Masako; Caballé-Serrano, Jordi; Bosshardt, Dieter D; Gruber, Reinhard; Buser, Daniel; Miron, Richard J

2016-07-04

The use of autogenous bone chips during guided bone regeneration procedures has remained the gold standard for bone grafting due to its excellent combination of osteoconduction, osteoinduction and osteogenesis. Recent protocols established by our group have characterized specific growth factors and cytokines released from autogenous bone that have the potential to be harvested and isolated into bone conditioned media (BCM). Due to the advantageous osteo-promotive properties of BCM, the aims of the present study was to pre-coat collagen barrier membranes with BCM and investigate its effect on osteoblast adhesion, proliferation and differentiation for possible future clinical use. Scanning electron microscopy (SEM) was first used to qualitative assess BCM protein accumulation on the surface of collagen membranes. Thereafter, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto BioGide porcine derived collagen barrier membranes (control) or barrier membranes pre-coated with BCM (test group). Control and BCM samples were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 5 days for osteoblast differentiation markers including Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Mineralization was further assessed with alizarin red staining at 14 days post seeding. SEM images demonstrated evidence of accumulated proteins found on the surface of collagen membranes following coating with BCM. Analysis of total cell numbers revealed that the additional pre-coating with BCM markedly increased cell attachment over 4 fold when compared to cells seeded on barrier membranes alone. No significant difference could be observed for cell proliferation at all time points. BCM significantly increased mRNA levels of osteoblast differentiation markers including ALP, OCN and BSP at 5 days post seeding. Furthermore, barrier membranes pre-coated with BCM demonstrated a 5-fold increase in alizarin red staining at 14 days. The results from the present study suggest that the osteoconductive properties of porcine-derived barrier membranes could be further improved by BCM by significantly increasing cell attachment, differentiation and mineralization of osteoblasts in vitro. Future animal testing is required to fully characterize the additional benefits of BCM for guided bone regeneration.

Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function.

PubMed

Tsukita, Sachiko; Katsuno, Tatsuya; Yamazaki, Yuji; Umeda, Kazuaki; Tamura, Atsushi; Tsukita, Shoichiro

2009-05-01

Tight junctions (TJs) create the primary permselective barrier to diffusion of solutes and ions through the paracellular pathway. The molecular architecture of TJs has gradually been unraveled in recent years, providing the basis for "barriology" (defined by Shoichiro Tsukita as the science of the barrier in multicellular organisms). Claudins are now considered to be the essential basic components of TJ strands, with which other integral membrane proteins, such as occludin, tricellulin, JAMs, and CAR, are associated. Peripherally associated scaffolding proteins are required for the organization of the integral membrane proteins. Among these, ZO-1, -2, and -3 have attracted a great deal of attention as TJ organizers, since ZO-1 (and in some cases, also ZO-2/3) was reported to be directly associated with claudins, occludin, and JAMs, as well as with AF-6/afadin and alpha-catenin. Here we summarize recent studies on ZO-1/2/3-deficiency in mice and cells, which have provided clear and important information regarding the functions of ZO-1/2/3 in vivo. In addition to the respective suppression of ZO-1/2/3 expression, simultaneous suppression of all three proteins has revealed the essential and nonessential in vivo roles of ZO-1/2 and ZO-3, respectively. ZO-3 shows an epithelial-specific TJ localization in a ZO-1/2-dependent fashion. ZO-1 and ZO-2 play pivotal roles in the final establishment of the belt-like adherens junctions (zonula adherens), followed by the formation of the belt-like TJs (zonula occludens) with paracellular barrier function, thereby providing the general basis for selective paracellular permeability in epithelial and endothelial cells.

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

PubMed Central

1982-01-01

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues. PMID:7142954

Scaffolding proteins in the development and maintenance of the epidermal permeability barrier.

PubMed

Crawford, Melissa; Dagnino, Lina

2017-10-02

The skin of mammals and other terrestrial vertebrates protects the organism against the external environment, preventing heat, water and electrolyte loss, as well as entry of chemicals and pathogens. Impairments in the epidermal permeability barrier function are associated with the genesis and/or progression of a variety of pathological conditions, including genetic inflammatory diseases, microbial and viral infections, and photodamage induced by UV radiation. In mammals, the outside-in epidermal permeability barrier is provided by the joint action of the outermost cornified layer, together with assembled tight junctions in granular keratinocytes found in the layers underneath. Tight junctions serve as both outside-in and inside-out barriers, and impede paracellular movements of ions, water, macromolecules and microorganisms. At the molecular level, tight junctions consist of integral membrane proteins that form an extracellular seal between adjacent cells, and associate with cytoplasmic scaffold proteins that serve as links with the actin cytoskeleton. In this review, we address the roles that scaffold proteins play specifically in the establishment and maintenance of the epidermal permeability barrier, and how various pathologies alter or impair their functions.

Ultrathin Transparent Membranes for Cellular Barrier and Co-Culture Models

PubMed Central

Carter, Robert N.; Casillo, Stephanie M.; Mazzocchi, Andrea R.; DesOrmeaux, Jon-Paul S.; Roussie, James A.; Gaborski, Thomas R.

2017-01-01

Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100–300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives. PMID:28140345

The role of barrier membranes for guided bone regeneration and restoration of large bone defects: current experimental and clinical evidence

PubMed Central

2012-01-01

Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery. Although there are several methods for bone reconstruction, they all have specific indications and limitations. The concept of using barrier membranes for restoration of bone defects has been developed in an effort to simplify their treatment by offering a sinlge-staged procedure. Research on this field of bone regeneration is ongoing, with evidence being mainly attained from preclinical studies. The purpose of this review is to summarize the current experimental and clinical evidence on the use of barrier membranes for restoration of bone defects in maxillofacial and orthopedic surgery. Although there are a few promising preliminary human studies, before clinical applications can be recommended, future research should aim to establish the 'ideal' barrier membrane and delineate the need for additional bone grafting materials aiming to 'mimic' or even accelerate the normal process of bone formation. Reproducible results and long-term observations with barrier membranes in animal studies, and particularly in large animal models, are required as well as well-designed clinical studies to evaluate their safety, efficacy and cost-effectiveness. PMID:22834465

Effect of porous xenographic bone graft with collagen barrier membrane on periodontal regeneration.

PubMed

Yamada, Satoru; Shima, Nobuhiro; Kitamura, Hidekazu; Sugito, Hiroki

2002-08-01

The purpose of this study was to investigate the effect of porous xenographic bone graft (Bio-Oss) with a collagen barrier membrane (Bio-Gide) on formation of new cementum and new bone in experimental intrabony defects of dogs. The intrabony defects were treated by either guided tissue regeneration with the collagen membrane (control group) or the collagen membrane with the porous bone mineral graft (experimental group). After 8 weeks, the animals were sacrificed and the tissues were histologically examined. New cementum with inserting collagen fibers was observed on the exposed surfaces in both groups. The amount of nevv bone was significantly greater in the group using the bone graft with the membrane than in the control group. The use of the collagen barrier membrane in combination with the porous bone graft material may enhance new bone and cementum formation.

Novel Nanocomposite Structures as Active and Passive Barrier Materials

DTIC Science & Technology

2010-06-01

during the course of this ARO-funded project. The development of a novel polymer material based on a diol-functionalized room-temperature ionic liquid ...material based on a diol-functionalized room- temperature ionic liquid (RTIL) monomer led to fabrication of membranes, which were tested for their...stimulant vapor. Technical Report A polymerizable room-temperature ionic liquid (4, Figure 1) was chosen as the starting material for making poly(RTIL

Structural basis for maintenance of bacterial outer membrane lipid asymmetry.

PubMed

Abellón-Ruiz, Javier; Kaptan, Shreyas S; Baslé, Arnaud; Claudi, Beatrice; Bumann, Dirk; Kleinekathöfer, Ulrich; van den Berg, Bert

2017-12-01

The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds 1 , 2 . The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet 1-3 . This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane 4 . The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C 5,6 , but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

Distinctive phytotoxic effects of Cd and Ni on membrane functionality.

PubMed

Sanz, Amparo; Llamas, Andreu; Ullrich, Cornelia I

2009-10-01

Metal ions essential for plant growth, such as Fe, Mn, Ni, Cu or Zn, are taken up by plants from the soil solution through metal transporters at the plasma membrane, mainly of the ZIP and Nramp families. These transport systems, however, can also give entry to other metals (Al, Cd, Hg, Pb). Non-nutritive elements, as well as the essential nutrients at higher than metabolic concentrations, can cause phytotoxicity. We have studied previously the effects of an essential (Ni) and a non essential (Cd) heavy metal on root cell plasma membranes, the first selective barrier encountered when entering the plant, using rice as model plant. Distinctive effects of Cd and Ni on membrane function (i.e., Em and membrane permeability) were observed in the short term. We have now confirmed the pattern of Em changes caused by Cd and Ni using barley roots and have also followed the effects of both metals in longer term in rice. Our data indicate that the distinct effects caused by Cd and Ni are due to differences in cellular responses, triggered when entering the cytoplasm (i.e., an efficient detoxifying mechanism for Cd), more than to different direct effects on membranes.

Combination of Collagen Barrier Membrane with Enamel Matrix Derivative-Liquid Improves Osteoblast Adhesion and Differentiation.

PubMed

Miron, Richard J; Fujioka-Kobayashi, Masako; Buser, Daniel; Zhang, Yufeng; Bosshardt, Dieter D; Sculean, Anton

Collagen barrier membranes were first introduced to regenerative periodontal and oral surgery to prevent fast ingrowing soft tissues (ie, epithelium and connective tissue) into the defect space. More recent attempts have aimed at combining collagen membranes with various biologics/growth factors to speed up the healing process and improve the quality of regenerated tissues. Recently, a new formulation of enamel matrix derivative in a liquid carrier system (Osteogain) has demonstrated improved physico-chemical properties for the adsorption of enamel matrix derivative to facilitate protein adsorption to biomaterials. The aim of this pioneering study was to investigate the use of enamel matrix derivative in a liquid carrier system in combination with collagen barrier membranes for its ability to promote osteoblast cell behavior in vitro. Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto porcine-derived collagen membranes alone (control) or porcine membranes + enamel matrix derivative in a liquid carrier system. Control and enamel matrix derivative-coated membranes were compared for cell recruitment and cell adhesion at 8 hours; cell proliferation at 1, 3, and 5 days; and real-time polymerase chain reaction (PCR) at 3 and 14 days for genes encoding Runx2, collagen1alpha2, alkaline phosphatase, and bone sialoprotein. Furthermore, alizarin red staining was used to investigate mineralization. A significant increase in cell adhesion was observed at 8 hours for barrier membranes coated with enamel matrix derivative in a liquid carrier system, whereas no significant difference could be observed for cell proliferation or cell recruitment. Enamel matrix derivative in a liquid carrier system significantly increased alkaline phosphatase mRNA levels 2.5-fold and collagen1alpha2 levels 1.7-fold at 3 days, as well as bone sialoprotein levels twofold at 14 days postseeding. Furthermore, collagen membranes coated with enamel matrix derivative in a liquid carrier system demonstrated a sixfold increase in alizarin red staining at 14 days when compared with collagen membrane alone. The combination of enamel matrix derivative in a liquid carrier system with a barrier membrane significantly increased cell attachment, differentiation, and mineralization of osteoblasts in vitro. Future animal testing is required to fully characterize the additional benefits of combining enamel matrix derivative in a liquid carrier system with a barrier membrane for guided bone or tissue regeneration.

Sterol Metabolism Disorders and Neurodevelopment--An Update

ERIC Educational Resources Information Center

Kanungo, Shibani; Soares, Neelkamal; He, Miao; Steiner, Robert D.

2013-01-01

Cholesterol has numerous quintessential functions in normal cell physiology, as well as in embryonic and postnatal development. It is a major component of cell membranes and myelin, and is a precursor of steroid hormones and bile acids. The development of the blood brain barrier likely around 12-18 weeks of human gestation makes the developing…

Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

PubMed

Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

2016-03-01

Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

Insight into podocyte differentiation from the study of human genetic disease: nail-patella syndrome and transcriptional regulation in podocytes.

PubMed

Morello, Roy; Lee, Brendan

2002-05-01

In recent years, our understanding of the molecular basis of kidney development has benefited from the study of rare genetic diseases affecting renal function. This has especially been the case with the differentiation of the highly specialized podocyte in the pathogenesis of human disorders and mouse phenotypes affecting the renal filtration barrier. This filtration barrier represents the end product of a complex series of signaling events that produce a tripartite structure consisting of interdigitating podocyte foot processes with intervening slit diaphragms, the glomerular basement membrane, and the fenestrated endothelial cell. Dysregulation of unique cytoskeletal and extracellular matrix proteins in genetic forms of nephrotic syndrome has shown how specific structural proteins contribute to podocyte function and differentiation. However, much less is known about the transcriptional determinants that both specify and maintain this differentiated cell. Our studies of a skeletal malformation syndrome, nail-patella syndrome, have shown how the LIM homeodomain transcription factor, Lmx1b, contributes to transcriptional regulation of glomerular basement membrane collagen expression by podocytes. Moreover, they raise intriguing questions about more global transcriptional regulation of podocyte morphogenesis.

Proton Transfer Dynamics at the Membrane/Water Interface: Dependence on the Fixed and Mobile pH Buffers, on the Size and Form of Membrane Particles, and on the Interfacial Potential Barrier

PubMed Central

Cherepanov, Dmitry A.; Junge, Wolfgang; Mulkidjanian, Armen Y.

2004-01-01

Crossing the membrane/water interface is an indispensable step in the transmembrane proton transfer. Elsewhere we have shown that the low dielectric permittivity of the surface water gives rise to a potential barrier for ions, so that the surface pH can deviate from that in the bulk water at steady operation of proton pumps. Here we addressed the retardation in the pulsed proton transfer across the interface as observed when light-triggered membrane proton pumps ejected or captured protons. By solving the system of diffusion equations we analyzed how the proton relaxation depends on the concentration of mobile pH buffers, on the surface buffer capacity, on the form and size of membrane particles, and on the height of the potential barrier. The fit of experimental data on proton relaxation in chromatophore vesicles from phototropic bacteria and in bacteriorhodopsin-containing membranes yielded estimates for the interfacial potential barrier for H+/OH− ions of ∼120 meV. We analyzed published data on the acceleration of proton equilibration by anionic pH buffers and found that the height of the interfacial barrier correlated with their electric charge ranging from 90 to 120 meV for the singly charged species to >360 meV for the tetra-charged pyranine. PMID:14747306

Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier.

PubMed

Bera, Swapna; Kar, Rajiv K; Mondal, Susanta; Pahan, Kalipada; Bhunia, Anirban

2016-09-06

Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQIKIWFQNRRMKWKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood-brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQIKIWFQNRRMKWKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6-Arg11, despite considerable conformational variation in different membrane models. In vivo data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders.

Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

PubMed

Zhang, Xi

2015-09-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

PubMed

Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

2014-01-09

The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

Nanomaterial interactions with biomembranes: Bridging the gap between soft matter models and biological context.

PubMed

Werner, Marco; Auth, Thorsten; Beales, Paul A; Fleury, Jean Baptiste; Höök, Fredrik; Kress, Holger; Van Lehn, Reid C; Müller, Marcus; Petrov, Eugene P; Sarkisov, Lev; Sommer, Jens-Uwe; Baulin, Vladimir A

2018-04-03

Synthetic polymers, nanoparticles, and carbon-based materials have great potential in applications including drug delivery, gene transfection, in vitro and in vivo imaging, and the alteration of biological function. Nature and humans use different design strategies to create nanomaterials: biological objects have emerged from billions of years of evolution and from adaptation to their environment resulting in high levels of structural complexity; in contrast, synthetic nanomaterials result from minimalistic but controlled design options limited by the authors' current understanding of the biological world. This conceptual mismatch makes it challenging to create synthetic nanomaterials that possess desired functions in biological media. In many biologically relevant applications, nanomaterials must enter the cell interior to perform their functions. An essential transport barrier is the cell-protecting plasma membrane and hence the understanding of its interaction with nanomaterials is a fundamental task in biotechnology. The authors present open questions in the field of nanomaterial interactions with biological membranes, including: how physical mechanisms and molecular forces acting at the nanoscale restrict or inspire design options; which levels of complexity to include next in computational and experimental models to describe how nanomaterials cross barriers via passive or active processes; and how the biological media and protein corona interfere with nanomaterial functionality. In this Perspective, the authors address these questions with the aim of offering guidelines for the development of next-generation nanomaterials that function in biological media.

Reduction of methanol crossover by thin cracked metal barriers at the interface between membrane and electrode in direct methanol fuel cells

NASA Astrophysics Data System (ADS)

Kim, Sungjun; Jang, Segeun; Kim, Sang Moon; Ahn, Chi-Yeong; Hwang, Wonchan; Cho, Yong-Hun; Sung, Yung-Eun; Choi, Mansoo

2017-09-01

This work reports the successful reduction in methanol crossover by creating a thin cracked metal barrier at the interface between a Nafion® membrane and an electrode in direct methanol fuel cells (DMFCs). The cracks are generated by simple mechanical stretching of a metal deposited Nafion® membrane as a result of the elastic mismatch between the two attached surfaces. The cracked metal barriers with varying strains (∼0.5 and ∼1.0) are investigated and successfully incorporated into the DMFC. Remarkably, the membrane electrode assembly with the thin metal crack exhibits comparable ohmic resistance as well as reduction of methanol crossover, which enhanced the device performance.

The Escherichia coli Phospholipase PldA Regulates Outer Membrane Homeostasis via Lipid Signaling.

PubMed

May, Kerrie L; Silhavy, Thomas J

2018-03-20

The outer membrane (OM) bilayer of Gram-negative bacteria is biologically unique in its asymmetrical organization of lipids, with an inner leaflet composed of glycerophospholipids (PLs) and a surface-exposed outer leaflet composed of lipopolysaccharide (LPS). This lipid organization is integral to the OM's barrier properties. Perturbations of the outer leaflet by antimicrobial peptides or defects in LPS biosynthesis or transport to the OM cause a compensatory flipping of PLs to the outer leaflet. As a result, lipid asymmetry is disrupted and OM integrity is compromised. Recently, we identified an Escherichia coli mutant that exhibits aberrant accumulation of surface PLs accompanied by a cellular increase in LPS production. Remarkably, the observed hyperproduction of LPS is PldA dependent. Here we provide evidence that the fatty acids generated by PldA at the OM are transported into the cytoplasm and simultaneously activated by thioesterification to coenzyme A (CoA) by FadD. The acyl-CoAs produced ultimately inhibit LpxC degradation by FtsH. The increased levels of LpxC, the enzyme that catalyzes the first committed step in LPS biosynthesis, increases the amount of LPS produced. Our data suggest that PldA acts as a sensor for lipid asymmetry in the OM. PldA protects the OM barrier by both degrading mislocalized PLs and generating lipid second messengers that enable long-distance signaling that prompts the cell to restore homeostasis at a distant organelle. IMPORTANCE The outer membrane of Gram-negative bacteria is an effective permeability barrier that protects the cell from toxic agents, including antibiotics. Barrier defects are often manifested by phospholipids present in the outer leaflet of this membrane that take up space normally occupied by lipopolysaccharide. We have discovered a signaling mechanism that operates across the entire cell envelope used by the cell to detect these outer membrane defects. A phospholipase, PldA, that functions to degrade these mislocalized phospholipids has a second, equally important function as a sensor. The fatty acids produced by hydrolysis of the phospholipids act as second messengers to signal the cell that more lipopolysaccharide is needed. These fatty acids diffuse across the periplasm and are transported into the cytoplasm by a process that attaches coenzyme A. The acyl-CoA molecule produces signals to inhibit the degradation of the critical enzyme LpxC by the ATP-dependent protease FtsH, increasing lipopolysaccharide production. Copyright © 2018 May and Silhavy.

Final Report - High Performance, Durable, Low Cost Membrane Electrode Assemblies for Transportation Applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinbach, Andrew

2017-05-31

The primary project objective was development of improved polymer electrolyte membrane fuel cell (PEMFC) membrane electrode assemblies (MEAs) which address the key DOE barriers of performance, durability and cost. Additional project objectives were to address commercialization barriers specific to MEAs comprising 3M nanostructured thin film (NSTF) electrodes, including a larger-than-acceptable sensitivity to operating conditions, an unexplained loss of rated power capability with operating time, and slow break-in conditioning. Significant progress was made against each of these barriers, and most DOE 2020 targets were met or substantially approached.

Effect of soaps and detergents on epidermal barrier function.

PubMed

Wolf, Ronni; Parish, Lawrence Charles

2012-01-01

The past decade has witnessed an explosion of new impartial information about the complex interaction of the skin with topically applied substances, including soaps and detergents. Despite of all these new data, our knowledge on the exact pathomechanism and molecular events leading to detergent-induced barrier dysfunction remains incomplete and the answers continue to elude us. The longtime prevailing opinion which contends that the damaging effect of soaps and detergents is related to their property to extract and remove useful intercellular lipids has mostly been abandoned. Although this effect might be involved in the damaging effect, it is definitely not the sole mechanism, nor, indeed, is it even the main one. Skin proteins damage, the interaction with keratins and their denaturation, swelling of cell membranes and collagen fibers, cytotoxicity expressed with cellular lysis are other important mechanisms. One proposed mechanism is that an initial stratum corneum hyper-hydration results from a continuous disruption of the secondary and tertiary structures of keratin protein by surfactants, exposing new water-binding sites, thereby increasing the hydration of the membrane. Following evaporation of excess water, the denatured keratin possesses a decreased water-binding capacity and decreased ability to function as a barrier. Recent studies have also emphasized the effects of detergents on lipid synthesis, on lipid-metabolizing enzymes and on keratinocyte differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Accumulation of phosphatidic acid increases vancomycin resistance in Escherichia coli.

PubMed

Sutterlin, Holly A; Zhang, Sisi; Silhavy, Thomas J

2014-09-01

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane, preventing entry of toxic molecules such as antibiotics. Mutations in lptD, the beta-barrel component of the LPS transport and assembly machinery, compromise LPS assembly and result in increased antibiotic sensitivity. Here, we report rare vancomycin-resistant suppressors that improve barrier function of a subset of lptD mutations. We find that all seven suppressors analyzed mapped to the essential gene cdsA, which is responsible for the conversion of phosphatidic acid to CDP-diacylglycerol in phospholipid biosynthesis. These cdsA mutations cause a partial loss of function and, as expected, accumulate phosphatidic acid. We show that this suppression is not confined to mutations that cause defects in outer membrane biogenesis but rather that these cdsA mutations confer a general increase in vancomycin resistance, even in a wild-type cell. We use genetics and quadrupole time of flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) to show that accumulation of phosphatidic acid by means other than cdsA mutations also increases resistance to vancomycin. We suggest that increased levels of phosphatidic acid change the physical properties of the outer membrane to impede entry of vancomycin into the periplasm, hindering access to its target, an intermediate required for the synthesis of the peptidoglycan cell wall. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Biomimetic carriers mimicking leukocyte plasma membrane to increase tumor vasculature permeability

NASA Astrophysics Data System (ADS)

Palomba, R.; Parodi, A.; Evangelopoulos, M.; Acciardo, S.; Corbo, C.; De Rosa, E.; Yazdi, I. K.; Scaria, S.; Molinaro, R.; Furman, N. E. Toledano; You, J.; Ferrari, M.; Salvatore, F.; Tasciotti, E.

2016-10-01

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

Negotiation of intracellular membrane barriers by TAT-modified gold nanoparticles.

PubMed

Krpetić, Zeljka; Saleemi, Samia; Prior, Ian A; Sée, Violaine; Qureshi, Rumana; Brust, Mathias

2011-06-28

This paper contributes to the debate on how nanosized objects negotiate membrane barriers inside biological cells. The uptake of peptide-modified gold nanoparticles by HeLa cells has been quantified using atomic emission spectroscopy. The TAT peptide from the HIV virus was singled out as a particularly effective promoter of cellular uptake. The evolution of the intracellular distribution of TAT-modified gold nanoparticles with time has been studied in detail by TEM and systematic image analysis. An unusual trend of particles disappearing from the cytosol and the nucleus and accumulating massively in vesicular bodies was observed. Subsequent release of the particles, both by membrane rupture and by direct transfer across the membrane boundary, was frequently found. Ultimately, near total clearing of particles from the cells occurred. This work provides support for the hypothesis that cell-penetrating peptides can enable small objects to negotiate membrane barriers also in the absence of dedicated transport mechanisms.

Hydrogen-permeable composite metal membrane and uses thereof

DOEpatents

Edlund, D.J.; Friesen, D.T.

1993-06-08

Various hydrogen production and hydrogen sulfide decomposition processes are disclosed that utilize composite metal membranes that contain an intermetallic diffusion barrier separating a hydrogen-permeable base metal and a hydrogen-permeable coating metal. The barrier is a thermally stable inorganic proton conductor.

The blood-cerebrospinal fluid barrier: structure and functional significance.

PubMed

Johanson, Conrad E; Stopa, Edward G; McMillan, Paul N

2011-01-01

The choroid plexus (CP) of the blood-CSF barrier (BCSFB) displays fundamentally different properties than blood-brain barrier (BBB). With brisk blood flow (10 × brain) and highly permeable capillaries, the human CP provides the CNS with a high turnover rate of fluid (∼400,000 μL/day) containing micronutrients, peptides, and hormones for neuronal networks. Renal-like basement membranes in microvessel walls and underneath the epithelium filter large proteins such as ferritin and immunoglobulins. Type IV collagen (α3, α4, and α5) in the subepithelial basement membrane confers kidney-like permselectivity. As in the glomerulus, so also in CP, the basolateral membrane utrophin A and colocalized dystrophin impart structural stability, transmembrane signaling, and ion/water homeostasis. Extensive infoldings of the plasma-facing basal labyrinth together with lush microvilli at the CSF-facing membrane afford surface area, as great as that at BBB, for epithelial solute and water exchange. CSF formation occurs by basolateral carrier-mediated uptake of Na+, Cl-, and HCO3-, followed by apical release via ion channel conductance and osmotic flow of water through AQP1 channels. Transcellular epithelial active transport and secretion are energized and channeled via a highly dense organelle network of mitochondria, endoplasmic reticulum, and Golgi; bleb formation occurs at the CSF surface. Claudin-2 in tight junctions helps to modulate the lower electrical resistance and greater permeability in CP than at BBB. Still, ratio analyses of influx coefficients (Kin) for radiolabeled solutes indicate that paracellular diffusion of small nonelectrolytes (e.g., urea and mannitol) through tight junctions is restricted; molecular sieving is proportional to solute size. Protein/peptide movement across BCSFB is greatly limited, occurring by paracellular leaks through incomplete tight junctions and low-capacity transcellular pinocytosis/exocytosis. Steady-state concentration ratios, CSF/plasma, ranging from 0.003 for IgG to 0.80 for urea, provide insight on plasma solute penetrability, barrier permeability, and CSF sink action to clear substances from CNS.

FOST 2 Upgrade with Hollow-Fiber CTA FO Module and Generation of Osmotic Agent for Microorganism Growth Studies

NASA Technical Reports Server (NTRS)

Parodi, Jurek; Mangado, Jaione Romero; Stefanson, Ofir; Flynn, Michael; Shaw, Hali; Beeler, David

2016-01-01

FOST 2 is an integrated membrane system that incorporates a forward osmosis subsystem and a reverse osmosis subsystem working in series. It has been designed as a post treatment system to process the effluent from the Membrane Aerated Biological Reactor developed at NASA Johnson Space Center and Texas Tech University. Its function is to remove dissolved solids residual such as ammonia and suspended solids, as well as to provide a physical barrier to microbial and viral contamination. A tubular CTA membrane module from HTI and a flat-sheet lipid-base membrane module from Porifera were integrated and tested on FOST 2 in the past, using both a bioreactor's effluent and greywater as the feed solution. This paper documents the performance of FOST 2 after its upgrade with a hollow-fiber CTA membrane module from Toyobo, treating real black-water to generate the osmotic agent solution necessary to conduct growth studies of genetically engineered microorganism for the Synthetic Biological Membrane project.

Transparent Metal-Organic Framework/Polymer Mixed Matrix Membranes as Water Vapor Barriers.

PubMed

Bae, Youn Jue; Cho, Eun Seon; Qiu, Fen; Sun, Daniel T; Williams, Teresa E; Urban, Jeffrey J; Queen, Wendy L

2016-04-27

Preventing the permeation of reactive molecules into electronic devices or photovoltaic modules is of great importance to ensure their life span and reliability. This work is focused on the formation of highly functioning barrier films based on nanocrystals (NCs) of a water-scavenging metal-organic framework (MOF) and a hydrophobic cyclic olefin copolymer (COC) to overcome the current limitations. Water vapor transmission rates (WVTR) of the films reveal a 10-fold enhancement in the WVTR compared to the substrate while maintaining outstanding transparency over most of the visible and solar spectrum, a necessary condition for integration with optoelectronic devices.

An alkaline phosphatase transport mechanism in the pathogenesis of Alzheimer's disease and neurodegeneration.

PubMed

Pike, Adrianne F; Kramer, Nynke I; Blaauboer, Bas J; Seinen, Willem; Brands, Ruud

2015-01-25

Systemic inflammation is associated with loss of blood-brain barrier integrity and neuroinflammation that lead to the exacerbation of neurodegenerative diseases. It is also associated specifically with the characteristic amyloid-β and tau pathologies of Alzheimer's disease. We have previously proposed an immunosurveillance mechanism for epithelial barriers involving negative feedback-regulated alkaline phosphatase transcytosis as an acute phase anti-inflammatory response that hangs in the balance between the resolution and the progression of inflammation. We now extend this model to endothelial barriers, particularly the blood-brain barrier, and present a literature-supported mechanistic explanation for Alzheimer's disease pathology with this system at its foundation. In this mechanism, a switch in the role of alkaline phosphatase from its baseline duties to a stopgap anti-inflammatory function results in the loss of alkaline phosphatase from cell membranes into circulation, thereby decreasing blood-brain barrier integrity and functionality. This occurs with impairment of both amyloid-β efflux and tau dephosphorylating activity in the brain as alkaline phosphatase is replenished at the barrier by receptor-mediated transport. We suggest systemic alkaline phosphatase administration as a potential therapy for the resolution of inflammation and the prevention of Alzheimer's disease pathology as well as that of other inflammation-related neurodegenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii].

PubMed

Abramycheva, N Iu; Govorun, V M

2000-01-01

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

PubMed

Shadforth, Audra M A; Suzuki, Shuko; Theodoropoulos, Christina; Richardson, Neil A; Chirila, Traian V; Harkin, Damien G

2017-06-01

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell ® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na + /K + -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Long-term drug release from electrospun fibers for in vivo inflammation prevention in the prevention of peritendinous adhesions.

PubMed

Hu, Changmin; Liu, Shen; Zhang, Yang; Li, Bin; Yang, Huilin; Fan, Cunyi; Cui, Wenguo

2013-07-01

Physical barriers such as electrospun fibrous membranes are potentially useful in preventing peritendinous adhesions after surgery. However, inflammatory responses caused by degradation of barrier materials remain a major challenge. This study aimed to fabricate electrospun composite fibrous membranes based on drug-loaded modified mesoporous silica (MMS) and poly (l-lactic acid) (PLLA). Using a co-solvent-based electrospinning method ibuprofen (IBU)-loaded MMS was successfully and uniformly encapsulated in the PLLA fibers. The electrospun PLLA-MMS-IBU composite fibrous membranes showed significantly lower initial burst release (6% release in the first 12h) compared with that of electrospun PLLA-IBU fibrous membranes (46% release in the first 12h) in in vitro release tests. Moreover, the release from PLLA-MMS-IBU was also for significantly longer than that from PLLA-IBU (100 vs. 20days). In animal studies both PLLA-IBU and PLLA-MMS-IBU showed improved anti-adhesion properties and anti-inflammatory effects compared with PLLA fibrous membrane alone 4weeks after implantation. Further, animals implanted with PLLA-MMS-IBU for 8weeks showed the lowest inflammation and best recovery compared with those implanted with PLLA-IBU and PLLA, most likely as a result of its long-term IBU release profile. Therefore, this study provides a platform technique for fabricating fibrous membranes with long-term sustained drug release characteristics which may function as a novel carrier for long-term anti-inflammation and anti-adhesion to prevent peritendinous adhesions. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Kinetics of human immunodeficiency virus budding and assembly

NASA Astrophysics Data System (ADS)

Zhang, Rui; Nguyen, Toan

2009-03-01

Human immunodeficiency virus (HIV) belongs to a large family of RNA viruses, retroviruses. Unlike budding of regular enveloped viruses, retroviruses bud concurrently with the assembly of retroviral capsids on the cell membrane. The kinetics of HIV (and other retroviruses) budding and assembly is therefore strongly affected by the elastic energy of the membrane and fundamentally different from regular viruses. The main result of this work shows that the kinetics is tunable from a fast budding process to a slow and effectively trapped partial budding process, by varying the attractive energy of retroviral proteins (call Gags), relative to the membrane elastic energy. When the Gag-Gag attraction is relatively high, the membrane elastic energy provides a kinetic barrier for the two pieces of the partial capsids to merge. This energy barrier determines the slowest step in the kinetics and the budding time. In the opposite limit, the membrane elastic energy provides not only a kinetic energy barrier, but a free energy barrier. The budding and assembly is effectively trapped at local free energy minimum, corresponding to a partially budded state. The time scale to escape from this metastable state is exponentially large. In both cases, our result fit with experimental measurements pretty well.

Pharmaceutical applications of cyclodextrins: effects on drug permeation through biological membranes.

PubMed

Loftsson, Thorsteinn; Brewster, Marcus E

2011-09-01

Cyclodextrins are useful solubilizing excipients that have gained currency in the formulator's armamentarium based on their ability to temporarily camouflage undesirable physicochemical properties. In this context cyclodextrins can increase oral bioavailability, stabilize compounds to chemical and enzymatic degradation and can affect permeability through biological membranes under certain circumstances. This latter property is examined herein as a function of the published literature as well as work completed in our laboratories.   Cyclodextrins can increase the uptake of drugs through biological barriers if the limiting barrier component is the unstirred water layer (UWL) that exists between the membrane and bulk water. This means that cyclodextrins are most useful when they interact with lipophiles in systems where such an UWL is present and contributes significantly to the barrier properties of the membrane. Furthermore, these principles are used to direct the optimal formulation of drugs in cyclodextrins. A second related critical success factor in the formulation of cyclodextrin-based drug product is an understanding of the kinetics and thermodynamics of complexation and the need to optimize the cyclodextrin amount and drug-to-cyclodextrin ratios. Drug formulations, especially those targeting compartments associated with limited dissolution (i.e. the eye, subcutaneous space, etc.), should be carefully designed such that the thermodynamic activity of the drug in the formulation is optimal meaning that there is sufficient cyclodextrin to solubilize the drug but not more than that. Increasing the cyclodextrin concentration decreases the formulation 'push' and may reduce the bioavailability of the system. A mechanism-based understanding of cyclodextrin complexation is essential for the appropriate formulation of contemporary drug candidates. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Osteoconductive Potential of Barrier NanoSiO2 PLGA Membranes Functionalized by Plasma Enhanced Chemical Vapour Deposition

PubMed Central

Terriza, Antonia; Vilches-Pérez, Jose I.; de la Orden, Emilio; Yubero, Francisco; Gonzalez-Caballero, Juan L.; González-Elipe, Agustin R.; Vilches, José; Salido, Mercedes

2014-01-01

The possibility of tailoring membrane surfaces with osteoconductive potential, in particular in biodegradable devices, to create modified biomaterials that stimulate osteoblast response should make them more suitable for clinical use, hopefully enhancing bone regeneration. Bioactive inorganic materials, such as silica, have been suggested to improve the bioactivity of synthetic biopolymers. An in vitro study on HOB human osteoblasts was performed to assess biocompatibility and bioactivity of SiO2 functionalized poly(lactide-co-glycolide) (PLGA) membranes, prior to clinical use. A 15 nm SiO2 layer was deposited by plasma enhanced chemical vapour deposition (PECVD), onto a resorbable PLGA membrane. Samples were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy, and infrared spectroscopy (FT-IR). HOB cells were seeded on sterilized test surfaces where cell morphology, spreading, actin cytoskeletal organization, and focal adhesion expression were assessed. As proved by the FT-IR analysis of samples, the deposition by PECVD of the SiO2 onto the PLGA membrane did not alter the composition and other characteristics of the organic membrane. A temporal and spatial reorganization of cytoskeleton and focal adhesions and morphological changes in response to SiO2 nanolayer were identified in our model. The novedous SiO2 deposition method is compatible with the standard sterilization protocols and reveals as a valuable tool to increase bioactivity of resorbable PLGA membranes. PMID:24883304

Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

PubMed Central

Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

2013-01-01

The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study.

PubMed

Lee, Nikki P Y; Cheng, C Yan

2003-07-01

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.

The urothelium: anatomy, review of the literature, perspectives for veterinary medicine.

PubMed

Arrighi, S

2015-03-01

Over time, much knowledge has been accumulated about the active role of the urothelium, principally in rodents and human. Far from being a mere passive barrier, this specialized epithelium can alter the ion and protein composition of the urine, is able to sense and respond to mechanical stimuli such as pressure, and react to mechanical stimuli by epithelial cell communication with the nervous system. Most of the specialized functions of the urothelium are linked to a number of morpho-physiologic properties exhibited by the superficial umbrella cells, including specialized membrane lipids, asymmetric unit membrane particles and a plasmalemma with stiff plaques which function as a barrier to most substances found in urine, thus protecting the underlying tissues. Moreover, the entire mucosa lining the low urinary tract, composed of urothelium and sub-urothelium, forms a functional transduction unit, able to respond to eso- and endogenous physical and chemical stimuli in a manner assuring an adequate functional response. This review will summarize the available information on each area of inquiry from a morpho-functional point of view. Possible considerations pertaining to species of veterinary interest are reviewed as well. The review was prepared consulting the electronic databases PubMed and Cab Abstracts and retrieving all pertinent reports and the relative reference lists, in order to identify any potential additional studies that could be included. Full-length research articles and thematic reviews were considered. Information on the urothelium of some domestic animal species was also included. Copyright © 2014 Elsevier GmbH. All rights reserved.

Liposomal membrane disruption by means of miniaturized dielectric-barrier discharge in air: liposome characterization

NASA Astrophysics Data System (ADS)

Svarnas, P.; Asimakoulas, L.; Katsafadou, M.; Pachis, K.; Kostazos, N.; Antimisiaris, S. G.

2017-08-01

The increasing interest of the plasma community in the application of atmospheric-pressure cold plasmas to bio-specimen treatment has led to the creation of the emerging field of plasma biomedicine. Accordingly, plasma setups based on dielectric-barrier discharges have already been widely tested for the inactivation of various cells. Most of these systems refer to the plasma jet concept where noble gases penetrate atmospheric air and are subjected to the influence of high electric fields, thus forming guided streamers. Following the original works of our group where liposomal membranes were proposed as models for studying the interaction between plasma jets and cells, we present herein a study on liposomal membrane disruption by means of miniaturized dielectric-barrier discharge running in atmospheric air. Liposomal membranes of various lipid compositions, lamellarities, and sizes are treated at different times. It is shown that the dielectric-barrier discharge of low mean power leads to efficient liposomal membrane disruption. The latter is achieved in a controllable manner and depends on liposome properties. Additionally, it is clearly demonstrated that liposomal membrane disruption takes place even after plasma extinction, i.e. during post-treatment, resembling thus an ‘apoptosis’ effect, which is well known today mainly for cell membranes. Thus, the adoption of the present concept would be beneficial for tailoring studies on plasma-treated cell-mimics. Finally, the liposome treatment is discussed with respect to possible physicochemical mechanisms and potential discharge modification due to the various compositions of the liquid electrode.

Motor Protein Myo1c Is a Podocyte Protein That Facilitates the Transport of Slit Diaphragm Protein Neph1 to the Podocyte Membrane ▿

PubMed Central

Arif, E.; Wagner, M. C.; Johnstone, D. B.; Wong, H. N.; George, B.; Pruthi, P. A.; Lazzara, M. J.; Nihalani, D.

2011-01-01

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function. PMID:21402783

Membrane tension and cytoskeleton organization in cell motility.

PubMed

Sens, Pierre; Plastino, Julie

2015-07-15

Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity.

The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

PubMed

Douglas, Lois M; Wang, Hong X; Konopka, James B

2013-11-26

Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane contributes to virulence, we analyzed a set of eight genes encoding MARVEL family proteins that are predicted to function in membrane organization. Interestingly, deletion of one gene, NCE102, caused a strong defect in formation of invasive hyphal growth in vitro and decreased virulence in mice. The nce102Δ mutant cells showed defects in actin organization that underlie the morphogenesis defect, since mutation of a known regulator of actin organization caused a similar defect. These studies identify a novel way in which the plasma membrane regulates the actin cytoskeleton and contributes to pathogenesis.

Thermodynamic analysis of membrane fouling in a submerged membrane bioreactor and its implications.

PubMed

Hong, Huachang; Peng, Wei; Zhang, Meijia; Chen, Jianrong; He, Yiming; Wang, Fangyuan; Weng, Xuexiang; Yu, Haiying; Lin, Hongjun

2013-10-01

The thermodynamic interactions between membrane and sludge flocs in a submerged membrane bioreactor (MBR) were investigated. It was found that Lewis acid-base (AB) interaction predominated in the total interactions. The interaction energy composition of membrane-sludge flocs combination was quite similar to that of membrane-bovine serum albumin (BSA) combination, indicating the critical role of proteins in adhesion process. Detailed analysis revealed the existence of a repulsive energy barrier in membrane-foulants interaction. Calculation results demonstrated that small flocs possessed higher attractive interaction energy per unit mass, and therefore adhered to membrane surface more easily as compared to large flocs. Meanwhile, initial sludge adhesion would facilitate the following adhesion due to the reduced repulsive energy barrier. Membrane with high electron donor surface tension component was a favor option for membrane fouling abatement. These findings offered new insights into membrane fouling, and also provided significant implications for fouling control in MBRs. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Blood-nerve barrier: structure and function].

PubMed

Kanda, Takashi

2011-06-01

The blood-nerve barrier (BNB) is a dynamic interface between the endoneurial microenvironment and surrounding extracellular space or blood contents, and is localized the innermost layer of multilayered ensheathing perineurium and endoneurial microvessels. Since the BNB is a key structure controlling the internal milieu of the peripheral nerve parenchyma, adequate understanding of the BNB is crucial for developing treatment strategies for human peripheral nervous system disorders, including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, and diabetic and various metabolic/toxic neuropathies. However, fewer studies have been conducted on the BNB, if we compare against the number of studies on the blood-brain barrier. This is because of the lack of adequate human cell lines originating from the BNB. In our laboratory, human immortal cell lines from the BNB, namely, the endothelial cell line and pericyte cell line, have recently been established and vigorous investigations of their biological and physiological properties are now underway. Pericytes constituting the BNB were found to possess robust ability of controlling BNB integrity via secretion of various cytokines and growth factors including bFGF, VEGF, GDNF, BDNF, and angiopoietin-1. Unknown soluble factors secreted by pericytes also contribute to the upregulation of claudin-5 in endothelial cells in the BNB and thus, strengthen the barrier function of the BNB. In diabetic neuropathy, pericytes were shown to regulate the vascular basement membrane, while AGEs were shown to induce basement membrane hypertrophy and disrupt the BNB by increasing the autocrine secretion of VEGF and TGF-beta from pericytes. In this review article, we discuss the macroscopic and microscopic anatomy of the human BNB as well as the molecular mechanisms of mononuclear cell infiltration across the BNB.

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets.

PubMed

Ryu, Seungmi; Yoo, Jin; Jang, Yeongseon; Han, Jin; Yu, Seung Jung; Park, Jooyeon; Jung, Seon Yeop; Ahn, Kyung Hyun; Im, Sung Gap; Char, Kookheon; Kim, Byung-Soo

2015-10-27

Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells.

Peptide-Lipid Interactions: Experiments and Applications

PubMed Central

Galdiero, Stefania; Falanga, Annarita; Cantisani, Marco; Vitiello, Mariateresa; Morelli, Giancarlo; Galdiero, Massimiliano

2013-01-01

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary. PMID:24036440

Enhanced methanol utilization in direct methanol fuel cell

DOEpatents

Ren, Xiaoming; Gottesfeld, Shimshon

2001-10-02

The fuel utilization of a direct methanol fuel cell is enhanced for improved cell efficiency. Distribution plates at the anode and cathode of the fuel cell are configured to distribute reactants vertically and laterally uniformly over a catalyzed membrane surface of the fuel cell. A conductive sheet between the anode distribution plate and the anodic membrane surface forms a mass transport barrier to the methanol fuel that is large relative to a mass transport barrier for a gaseous hydrogen fuel cell. In a preferred embodiment, the distribution plate is a perforated corrugated sheet. The mass transport barrier may be conveniently increased by increasing the thickness of an anode conductive sheet adjacent the membrane surface of the fuel cell.

JAM related proteins in mucosal homeostasis and inflammation

PubMed Central

Luissint, Anny-Claude; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include Junctional Adhesion Molecules (JAMs) that belong to the CTX (Cortical Thymocyte marker for Xenopus) family of proteins. JAM family encompasses three classical members (JAM-A, -B and –C) and related molecules including JAM4, JAM-Like protein (JAM-L), Coxsackie and Adenovirus Receptor (CAR), CAR-Like Membrane Protein (CLMP) and Endothelial cell-Selective Adhesion Molecule (ESAM). JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation. PMID:24667924

Electrical measurement of the hydration state of the skin surface in vivo.

PubMed

Tagami, H

2014-09-01

Healthy skin surface is smooth and soft, because it is covered by the properly hydrated stratum corneum (SC), an extremely thin and soft barrier membrane produced by the underlying normal epidermis. By contrast, the skin surfaces covering pathological lesions exhibit dry and scaly changes and the SC shows poor barrier function. The SC barrier function has been assessed in vivo by instrumentally measuring transepidermal water loss (TEWL). However, there was a lack of any appropriate method for evaluating the hydration state of the skin surface in vivo until 1980 when we reported the feasibility of employing high-frequency conductance or capacitance to evaluate it quickly and accurately. With such measurements, we can assess easily the moisturizing efficacy of various topical agents in vivo as well as the distribution pattern of water in the SC by combining it with a serial tape-stripping procedure of the skin surface. © 2014 The Author BJD © 2014 British Association of Dermatologists.

Insight into proton transfer in phosphotungstic acid functionalized mesoporous silica-based proton exchange membrane fuel cells.

PubMed

Zhou, Yuhua; Yang, Jing; Su, Haibin; Zeng, Jie; Jiang, San Ping; Goddard, William A

2014-04-02

We have developed for fuel cells a novel proton exchange membrane (PEM) using inorganic phosphotungstic acid (HPW) as proton carrier and mesoporous silica as matrix (HPW-meso-silica) . The proton conductivity measured by electrochemical impedance spectroscopy is 0.11 S cm(-1) at 90 °C and 100% relative humidity (RH) with a low activation energy of ∼14 kJ mol(-1). In order to determine the energetics associated with proton migration within the HPW-meso-silica PEM and to determine the mechanism of proton hopping, we report density functional theory (DFT) calculations using the generalized gradient approximation (GGA). These DFT calculations revealed that the proton transfer process involves both intramolecular and intermolecular proton transfer pathways. When the adjacent HPWs are close (less than 17.0 Å apart), the calculated activation energy for intramolecular proton transfer within a HPW molecule is higher (29.1-18.8 kJ/mol) than the barrier for intermolecular proton transfer along the hydrogen bond. We find that the overall barrier for proton movement within the HPW-meso-silica membranes is determined by the intramolecular proton transfer pathway, which explains why the proton conductivity remains unchanged when the weight percentage of HPW on meso-silica is above 67 wt %. In contrast, the activation energy of proton transfer on a clean SiO2 (111) surface is computed to be as high as ∼40 kJ mol(-1), confirming the very low proton conductivity on clean silica surfaces observed experimentally.

A strain-controlled C2N monolayer membrane for gas separation in PEMFC application

NASA Astrophysics Data System (ADS)

Deng, Shengwei; Hu, Hui; Zhuang, Guilin; Zhong, Xing; Wang, Jianguo

2018-05-01

Ultrathin membranes with controllable pore sizes have great potential to realize high-selectivity gas separation at low energy cost, especially for those mixtures with narrow size distributions. Using a combination of van der Waals-corrected density functional theory (DFT) calculations and molecular dynamics (MD) simulation, we examine the separation ability of biaxial stretched monolayer C2N nanosheets which is applied to the O2 separation from CO/CO2/O2 mixtures in the cathode of proton exchange membrane fuel cells (PEMFC). The DFT calculations show that the diffusion energy barrier for molecules passing through the membrane followed by CO, CO2 and O2 in descending order, and an overall decrease of energy barriers due to the widen the pore size is observed with the increase of applied strains. Furthermore, MD results show that the nanosheet can effectively purify O2 from CO2 and CO with a strain from 8% to 10%. It confirms that the selectivity is determined by the electronic structure related interaction in addition to the kinetic diameter of individual molecules. The O2 permeability is improved progressively with further increase of strain, and small amount of CO2 begins to permeate through the nanosheet at relatively large strain, while the excellent CO isolation is not compromised until the theoretical maximum strain.

String Vessel Formation is Increased in the Brain of Parkinson Disease.

PubMed

Yang, Panzao; Pavlovic, Darja; Waldvogel, Henry; Dragunow, Mike; Synek, Beth; Turner, Clinton; Faull, Richard; Guan, Jian

2015-01-01

String vessels are collapsed basement membrane without endothelium and have no function in circulation. String vessel formation contributes to vascular degeneration in Alzheimer disease. By comparing to age-matched control cases we have recently reported endothelial degeneration in brain capillaries of human Parkinson disease (PD). Current study evaluated changes of basement membrane of capillaries, string vessel formation and their association with astrocytes, blood-brain-barrier integrity and neuronal degeneration in PD. Brain tissue from human cases of PD and age-matched controls was used. Immunohistochemical staining for collagen IV, GFAP, NeuN, tyrosine hydroxylase, fibrinogen and Factor VIII was evaluated by image analysis in the substantia nigra, caudate nucleus and middle frontal gyrus. While the basement-membrane-associated vessel density was similar between the two groups, the density of string vessels was significantly increased in the PD cases, particularly in the substantia nigra. Neuronal degeneration was found in all brain regions. Astrocytes and fibrinogen were increased in the caudate nuclei of PD cases compared with control cases. Endothelial degeneration and preservation of basement membrane result in an increase of string vessel formation in PD. The data may suggest a possible role for cerebral hypoperfusion in the neuronal degeneration characteristic of PD, which needs further investigation. Elevated astrocytosis in the caudate nucleus of PD cases could be associated with disruption of the blood-brain barrier in this brain region.

Simulations of skin barrier function: free energies of hydrophobic and hydrophilic transmembrane pores in ceramide bilayers.

PubMed

Notman, Rebecca; Anwar, Jamshed; Briels, W J; Noro, Massimo G; den Otter, Wouter K

2008-11-15

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tail-vapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening.

Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

PubMed Central

Notman, Rebecca; Anwar, Jamshed; Briels, W. J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tail-vapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening. PMID:18708461

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

PubMed

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

How do biological systems discriminate among physically similar ions?

PubMed

Diamond, J M

1975-10-01

This paper reviews the history of understanding how biological systems can discriminate so strikingly among physically similar ions, especially alkali cations. Appreciation of qualitative regularities ("permitted sequences") and quantitative regularities ("selectivity isotherms") in ion selectivity grew first from studies of ion exchangers and glass electrodes, then of biological systems such as enzymes and cell membranes, and most recently of lipid bilayers doped with model pores and carriers. Discrimination of ions depends on both electrostatic and steric forces. "Black-box" studies on intact biological membranes have in some cases yielded molecular clues to the structure of the actual biological pores and carriers. Major current problems involve the extraction of these molecules; how to do it, what to do when it is achieved, and how (and if) it is relevant to the central problems of membrane function. Further advances are expected soon from studies of rate barriers within membranes, of voltage-dependent ("excitable") conducting channels, and of increasingly complex model systems and biological membranes.

Mapping the energy landscape for second-stage folding of a single membrane protein

PubMed Central

Min, Duyoung; Jefferson, Robert E; Bowie, James U; Yoon, Tae-Young

2016-01-01

Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ~3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. PMID:26479439

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

46 CFR 154.431 - Model test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 5 2010-10-01 2010-10-01 false Model test. 154.431 Section 154.431 Shipping COAST GUARD... Model test. (a) The primary and secondary barrier of a membrane tank, including the corners and joints...(c). (b) Analyzed data of a model test for the primary and secondary barrier of the membrane tank...

General Protein Diffusion Barriers create Compartments within Bacterial Cells

PubMed Central

Schlimpert, Susan; Klein, Eric A.; Briegel, Ariane; Hughes, Velocity; Kahnt, Jörg; Bolte, Kathrin; Maier, Uwe G.; Brun, Yves V.; Jensen, Grant J.; Gitai, Zemer; Thanbichler, Martin

2013-01-01

SUMMARY In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell cycle-dependent manner. Their presence is critical for cellular fitness, as they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins. PMID:23201141

Fabrication of Novel Hydrogel with Berberine-Enriched Carboxymethylcellulose and Hyaluronic Acid as an Anti-Inflammatory Barrier Membrane

PubMed Central

Huang, Yu-Chih; Huang, Kuen-Yu; Yang, Bing-Yuan

2016-01-01

An antiadhesion barrier membrane is an important biomaterial for protecting tissue from postsurgical complications. However, there is room to improve these membranes. Recently, carboxymethylcellulose (CMC) incorporated with hyaluronic acid (HA) as an antiadhesion barrier membrane and drug delivery system has been reported to provide excellent tissue regeneration and biocompatibility. The aim of this study was to fabricate a novel hydrogel membrane composed of berberine-enriched CMC prepared from bark of the P. amurense tree and HA (PE-CMC/HA). In vitro anti-inflammatory properties were evaluated to determine possible clinical applications. The PE-CMC/HA membranes were fabricated by mixing PE-CMC and HA as a base with the addition of polyvinyl alcohol to form a film. Tensile strength and ultramorphology of the membrane were evaluated using a universal testing machine and scanning electron microscope, respectively. Berberine content of the membrane was confirmed using a UV-Vis spectrophotometer at a wavelength of 260 nm. Anti-inflammatory property of the membrane was measured using a Griess reaction assay. Our results showed that fabricated PE-CMC/HA releases berberine at a concentration of 660 μg/ml while optimal plasticity was obtained at a 30 : 70 PE-CMC/HA ratio. The berberine-enriched PE-CMC/HA had an inhibited 60% of inflammation stimulated by LPS. These results suggest that the PE-CMC/HA membrane fabricated in this study is a useful anti-inflammatory berberine release system. PMID:28119926

Atomistic and continuum scale modeling of functionalized graphyne membranes for water desalination.

PubMed

Raju, Muralikrishna; Govindaraju, Pavan B; van Duin, Adri C T; Ihme, Matthias

2018-02-22

Recent theoretical and experimental studies reported ultra-high water permeability and salt rejection in nanoporous single-layer graphene. However, creating and controlling the size and distribution of nanometer-scale pores pose significant challenges to application of these membranes for water desalination. Graphyne and hydrogenated graphyne have tremendous potential as ultra-permeable membranes for desalination and wastewater reclamation due to their uniform pore-distribution, atomic thickness and mechano-chemical stability. Using molecular dynamics (MD) simulations and upscale continuum analysis, the desalination performance of bare and hydrogenated α-graphyne and γ-{2,3,4}-graphyne membranes is evaluated as a function of pore size, pore geometry, chemical functionalization and applied pressure. MD simulations show that pores ranging from 20 to 50 Å 2 reject in excess of 90% of the ions for pressures up to 1 GPa. Water permeability is found to range up to 85 L cm -2 day -1 MPa -1 , which is up to three orders of magnitude larger than commercial seawater reverse osmosis (RO) membranes and up to ten times that of nanoporous graphene. Pore chemistry, functionalization and geometry are shown to play a critical role in modulating the water flux, and these observations are explained by water velocity, density, and energy barriers in the pores. The atomistic scale investigations are complemented by upscale continuum analysis to examine the performance of these membranes in application to cross-flow RO systems. This upscale analysis, however, shows that the significant increase in permeability, observed from MD simulations, does not fully translate to current RO systems due to transport limitations. Nevertheless, upscale calculations predict that the higher permeability of graphyne membranes would allow up to six times higher permeate recovery or up to 6% less energy consumption as compared to thin-film composite membranes at currently accessible operating conditions. Significantly higher energy savings and permeate recovery can be achieved if higher feed-flow rates can be realized.

Mechanism for degradation of Nafion in PEM fuel cells from quantum mechanics calculations.

PubMed

Yu, Ted H; Sha, Yao; Liu, Wei-Guang; Merinov, Boris V; Shirvanian, Pezhman; Goddard, William A

2011-12-14

We report results of quantum mechanics (QM) mechanistic studies of Nafion membrane degradation in a polymer electrolyte membrane (PEM) fuel cell. Experiments suggest that Nafion degradation is caused by generation of trace radical species (such as OH(●), H(●)) only when in the presence of H(2), O(2), and Pt. We use density functional theory (DFT) to construct the potential energy surfaces for various plausible reactions involving intermediates that might be formed when Nafion is exposed to H(2) (or H(+)) and O(2) in the presence of the Pt catalyst. We find a barrier of 0.53 eV for OH radical formation from HOOH chemisorbed on Pt(111) and of 0.76 eV from chemisorbed OOH(ad), suggesting that OH might be present during the ORR, particularly when the fuel cell is turned on and off. Based on the QM, we propose two chemical mechanisms for OH radical attack on the Nafion polymer: (1) OH attack on the S-C bond to form H(2)SO(4) plus a carbon radical (barrier: 0.96 eV) followed by decomposition of the carbon radical to form an epoxide (barrier: 1.40 eV). (2) OH attack on H(2) crossover gas to form hydrogen radical (barrier: 0.04 eV), which subsequently attacks a C-F bond to form HF plus carbon radicals (barrier as low as 1.00 eV). This carbon radical can then decompose to form a ketone plus a carbon radical with a barrier of 0.86 eV. The products (HF, OCF(2), SCF(2)) of these proposed mechanisms have all been observed by F NMR in the fuel cell exit gases along with the decrease in pH expected from our mechanism. © 2011 American Chemical Society

Influence of cell shape, inhomogeneities and diffusion barriers in cell polarization models

NASA Astrophysics Data System (ADS)

Giese, Wolfgang; Eigel, Martin; Westerheide, Sebastian; Engwer, Christian; Klipp, Edda

2015-12-01

In silico experiments bear the potential for further understanding of biological transport processes by allowing a systematic modification of any spatial property and providing immediate simulation results. Cell polarization and spatial reorganization of membrane proteins are fundamental for cell division, chemotaxis and morphogenesis. We chose the yeast Saccharomyces cerevisiae as an exemplary model system which entails the shuttling of small Rho GTPases such as Cdc42 and Rho, between an active membrane-bound form and an inactive cytosolic form. We used partial differential equations to describe the membrane-cytosol shuttling of proteins. In this study, a consistent extension of a class of 1D reaction-diffusion systems into higher space dimensions is suggested. The membrane is modeled as a thin layer to allow for lateral diffusion and the cytosol is modeled as an enclosed volume. Two well-known polarization mechanisms were considered. One shows the classical Turing-instability patterns, the other exhibits wave-pinning dynamics. For both models, we investigated how cell shape and diffusion barriers like septin structures or bud scars influence the formation of signaling molecule clusters and subsequent polarization. An extensive set of in silico experiments with different modeling hypotheses illustrated the dependence of cell polarization models on local membrane curvature, cell size and inhomogeneities on the membrane and in the cytosol. In particular, the results of our computer simulations suggested that for both mechanisms, local diffusion barriers on the membrane facilitate Rho GTPase aggregation, while diffusion barriers in the cytosol and cell protrusions limit spontaneous molecule aggregations of active Rho GTPase locally.

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin*

PubMed Central

Schurek, Eva-Maria; Völker, Linus A.; Tax, Judit; Lamkemeyer, Tobias; Rinschen, Markus M.; Ungrue, Denise; Kratz, John E.; Sirianant, Lalida; Kunzelmann, Karl; Chalfie, Martin; Schermer, Bernhard; Benzing, Thomas; Höhne, Martin

2014-01-01

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier. PMID:24596097

Amphoteric Ion-Exchange Membranes with Significantly Improved Vanadium Barrier Properties for All-Vanadium Redox Flow Batteries.

PubMed

Nibel, Olga; Rojek, Tomasz; Schmidt, Thomas J; Gubler, Lorenz

2017-07-10

All-vanadium redox flow batteries (VRBs) have attracted considerable interest as promising energy-storage devices that can allow the efficient utilization of renewable energy sources. The membrane, which separates the porous electrodes in a redox flow cell, is one of the key components in VRBs. High rates of crossover of vanadium ions and water through the membrane impair the efficiency and capacity of a VRB. Thus, membranes with low permeation rate of vanadium species and water are required, also characterized by low resistance and stability in the VRB environment. Here, we present a new design concept for amphoteric ion-exchange membranes, based on radiation-induced grafting of vinylpyridine into an ethylene tetrafluoroethylene base film and a two-step functionalization to introduce cationic and anionic exchange sites, respectively. During long-term cycling, redox flow cells containing these membranes showed higher efficiency, less pronounced electrolyte imbalance, and significantly reduced capacity decay compared to the cells with the benchmark material Nafion 117. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermodynamic assessment of adsorptive fouling with the membranes modified via layer-by-layer self-assembly technique.

PubMed

Shen, Liguo; Cui, Xia; Yu, Genying; Li, Fengquan; Li, Liang; Feng, Shushu; Lin, Hongjun; Chen, Jianrong

2017-05-15

In this study, polyvinylidene fluoride (PVDF) microfiltration membrane was coated by dipping the membrane alternatingly in solutions of the polyelectrolytes (poly-diallyldimethylammonium chloride (PDADMAC) and polystyrenesulfonate (PSS)) via layer-by-layer (LBL) self-assembly technique to improve the membrane antifouling ability. Filtration experiments showed that, sludge cake layer on the coated membrane could be more easily washed off, and moreover, the remained flux ratio (RFR) of the coated membrane was obviously improved as compared with the control membrane. Characterization of the membranes showed that a polyelectrolyte layer was successfully coated on the membrane surfaces, and the hydrophilicity, surface charge and surface morphology of the coated membrane were changed. Based on the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) approaches, quantification of interfacial interactions between foulants and membranes in three different scenarios was achieved. It was revealed that there existed a repulsive energy barrier when a particle foulant adhered to membrane surface, and the enhanced electrostatic double layer (EL) interaction and energy barrier should be responsible for the improved antifouling ability of the coated membrane. This study provided a combined solution to membrane modification and interaction energy evaluation related with membrane fouling simultaneously. Copyright © 2017 Elsevier Inc. All rights reserved.

[Blood-nerve barrier and peripheral nerve regeneration].

PubMed

Kanda, Takashi

2013-01-01

Blood-nerve barrier (BNB) restricts the movement of soluble mediators and leukocytes from the blood contents to the peripheral nervous system (PNS) parenchyma and thus maintains the endoneurial homeostasis. However, it interferes the supply of various neurotrophic factors from the blood constituents and stops the drainage of toxic substances out of the PNS parenchyma, resulting in the inhibition of peripheral nerve regeneration. If the manipulation of BNB function is possible, regeneration of peripheral nerve may be facilitated via the alteration of peripheral nerve microenvironment and ample supply of neurotrophic substances. A possible method to manipulate the BNB for therapeutic purposes is to modify the endothelial function using siRNAs, oligonucleotides and virus vectors. Another possible method is to modify BNB pericytes: small hydrophobic substances that can reach the pericyte membrane through the endothelial monolayer and strengthen the pericytic activity, including the release of various cytokines/chemokines that influence endothelial function, may also be useful as drug candidates to control the BNB function.

The effects of combined liquid and membrane barriers in prevention of post-operative intra-abdominal adhesions after experimental jejunal anastomosis in dogs.

PubMed

el-Ghoul, W

2005-01-01

The study was carried out on 40 apparently clinical healthy dogs classified into 5 groups of 8 dogs each. Adhesion was experimentally induced by transsection and reanastomosis of jejunum. In the control group the site of anastomosis and abdominal cavity was lavaged with 250 ml saline solution. In group two lavage was done with 250 ml of a liquid barrier composed of a combination of high molecular weight solution (1% sodium carboxymethylcellulose) as a carrier, non-steroidal anti-inflammatory drug (Piroxecam), broad spectrum antibiotic (Cephalosporin), anticoagulant (Heparin) and antioxidant (0.5% methylene blue). In group three the anastomosis site was covered with a sodium hyalouronate/carboxymethylcellulose bioresorbable membrane (Seprafilm). In group four a natural biocompatible collagen sheet (VET BIO SIS T) was applied on the anastomosis site. In group five the abdominal cavity was lavaged with 250 ml liquid barrier and the anastomosis site was covered by either Seprafilm membrane or VET BIO SIS T sheet. At the fourteen day after operation, adhesion was assessed by ultrasonography after instillation of 1000 ml of physiological saline solution into the abdominal cavity. The dogs were sacrificed and an autopsy examination was carried out with the attention to the number, density and site of the adhesion formation. The results revealed that all the control dogs and some dogs in the treatment groups had positive ultrasonographic findings. Transabdominal sonogram clearly showed echogenic bands floating in the abdominal cavity and echogenic masses in more serious subjects. Necropsy examination showed that all the control dogs had intra-abdominal adhesions (8 of 8 dogs) and treatment with liquid barrier (4 of 8 dogs), seprafilm membrane barrier (3 of 8 dogs), VET BIO SIS T sheet barrier (4 of 8 dogs) and combination of fluid and membrane barrier groups (4 of 8 dogs) significantly (p < 0.05) reduced the incidence of adhesion formation. The adhesion severity in the four treated groups was significantly (p < 0.05) decreased compared with the control group as shown by both ultrasonography and necropsy examination scores. In conclusion the suggested hypothesis is more or less positive and the combined liquid and membrane barriers might be an effective way to decrease intra-abdominal adhesion formation, and the ultrasonography is a useful tool to diagnose intra-abdominal adhesion, and their applications might be valuable to the clinical settings.

Enhanced surface hydrophilicity of thin-film composite membranes for nanofiltration: an experimental and DFT study.

PubMed

Lv, Zhiwei; Hu, Jiahui; Zhang, Xuan; Wang, Lianjun

2015-10-07

In the current study, thin-film composite (TFC) nanofiltration membranes desirable for water softening were successfully developed through interfacial polymerization using N-(2-hydroxyethyl)ethylenediamine (HEDA) as the amine monomer in the aqueous phase. The hydrophilicity of the membrane surface was greatly enhanced with the introduction of the residual hydroxyl groups during the fabrication process. The TFC membranes possessed a permeate flux of 15.8 L m(-2) h(-1) under 0.6 MPa, with a rejection of 85.9%, 73.8%, and 99.8% for Na2SO4, MgSO4 and Congo red, respectively. The interplays of the solvent, solute and polymer matrix on the separation performance were investigated by means of the solubility parameter study. Moreover, density functional theory was employed to calculate the Fukui function by the Hirshfeld charge, which gave the global and local softness values to predict the reactivity of the atomic sites in the HEDA molecule. The findings of this study support the possible forming mechanism of the barrier layer for the first time. The TFC membrane was found to be stable and displayed good separation ability over a week-long filtration process. The combined results of this work suggest that these HEDA/TMC TFC nanofiltration membranes are promising candidates for various applications, such as desalination and dye removal from wastewater.

Blood-urine barrier formation in mouse urinary bladder development.

PubMed

Jezernik, K; Pipan, N

1993-04-01

Formation of the blood-urine permeability barrier in differentiating mouse transitional urothelium was studied. It was established that the development of superficial cell barrier is a two-phase process: beginning with formation of the tight junctions, followed by formation of fusiform vesicles and asymmetric apical plasma membranes. Fusiform vesicles differentiate during days 15 and 17 of gestation and fuse with the apical plasmalemma. Thus a thick membrane is formed before the excretion of hypertonic urine into the embryonic bladder. Through some degenerative superficial cells slough between fetal day 17 and the day of birth, the bladder epithelium in mice does not lack an effective permeability barrier.

Possible Involvement of Tight Junctions, Extracellular Matrix and Nuclear Receptors in Epithelial Differentiation

PubMed Central

Ichikawa-Tomikawa, Naoki; Sugimoto, Kotaro; Satohisa, Seiro; Nishiura, Keisuke; Chiba, Hideki

2011-01-01

Tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane. They consist of four kinds of transmembrane proteins (occludin, claudins, junctional adhesion molecules, and tricellulin) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function. The mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases. In this paper, we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions, extracellular matrix, and nuclear receptors in epithelial differentiation. PMID:22162632

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

PubMed Central

Maxson, Michelle E; Naj, Xenia; O'Meara, Teresa R; Plumb, Jonathan D; Cowen, Leah E

2018-01-01

Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae. PMID:29553370

Tests of potential functional barriers for laminated multilayer food packages. Part II: Medium molecular weight permeants.

PubMed

Simal-Gándara, J; Sarria-Vidal, M; Rijk, R

2000-09-01

Experiments were performed to characterize the kinetics of the permeation of different medium molecular weight model permeants: bisphenol A, warfarin and anthracene, from liquid paraffin, through a surrogate potential functional barrier (25 microns-thick orientated polypropylene--OPP) into the food simulants olive oil and 3% (w/v) acetic acid. The characterization of permeation kinetics generally observed the permeation models previously reported to explain the experimental permeation results obtained for a low molecular weight group of model permeants. In general, the model permeants exhibited behaviour consistent with their relative molecular weights with respect to (a) the time taken to attain steady-state permeation into the food simulant in which they were more soluble, (b) their subsequent steady-state permeation rates, and (c) their partition between liquid paraffin and the OPP membrane.

Role of the α clamp in the protein translocation mechanism of anthrax toxin

PubMed Central

Brown, Michael J.; Thoren, Katie L.; Krantz, Bryan A.

2015-01-01

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor (LF), and edema factor (EF). Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds LF and EF and translocates them into the host cytosol. Translocation is driven by the proton motive force, comprised of the chemical potential, the proton-gradient (ΔpH), and the membrane potential (ΔΨ). A crystal structure of the lethal toxin core complex revealed an “α clamp” structure that binds to substrate helices nonspecifically. Here we test the hypothesis that through the recognition of unfolding helical structure the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation. PMID:26344833

Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells.

PubMed

Wu, Xin-Sheng; Elias, Sharon; Liu, Huisheng; Heureaux, Johanna; Wen, Peter J; Liu, Allen P; Kozlov, Michael M; Wu, Ling-Gang

2017-12-05

Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis. Published by Elsevier Inc.

Site-specific distribution of claudin-based paracellular channels with roles in biological fluid flow and metabolism.

PubMed

Tanaka, Hiroo; Tamura, Atsushi; Suzuki, Koya; Tsukita, Sachiko

2017-10-01

The claudins are a family of membrane proteins with at least 27 members in humans and mice. The extracellular regions of claudin proteins play essential roles in cell-cell adhesion and the paracellular barrier functions of tight junctions (TJs) in epithelial cell sheets. Furthermore, the extracellular regions of some claudins function as paracellular channels in the paracellular barrier that allow the selective passage of water, ions, and/or small organic solutes across the TJ in the extracellular space. Structural analyses have revealed a common framework of transmembrane, cytoplasmic, and extracellular regions among the claudin-based paracellular barriers and paracellular channels; however, differences in the claudins' extracellular regions, such as their charges and conformations, determine their properties. Among the biological systems that involve fluid flow and metabolism, it is noted that hepatic bile flow, renal Na + reabsorption, and intestinal nutrient absorption are dynamically regulated via site-specific distributions of paracellular channel-forming claudins in tissue. Here, we focus on how site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine. © 2017 New York Academy of Sciences.

Brain delivery of proteins via their fatty acid and block copolymer modifications

PubMed Central

Yi, Xiang; Kabanov, Alexander V.

2014-01-01

It is well known that hydrophobic small molecules penetrate cell membranes better than hydrophilic molecules. Amphiphilic molecules that dissolve both in lipid and aqueous phases are best suited for membrane transport. Transport of biomacromolecules across physiological barriers, e.g. the blood-brain barrier, is greatly complicated by the unique structure and function of such barriers. Two decades ago we adopted a simple philosophy that to increase protein delivery to the brain one needs to modify this protein with hydrophobic moieties. With this general idea we began modifying proteins (antibodies, enzymes, hormones, etc.) with either hydrophobic fatty acid residues or amphiphilic block copolymer moieties, such as poy(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) and more recently, poly(2-oxasolines). This simple approach has resulted in impressive successes in CNS drug delivery. We present a retrospective overview of these works initiated in the Soviet Union in 1980s, and then continued in the United States and other countries. Notably some of the early findings were later corroborated by brain pharmacokinetic data. Industrial development of several drug candidates employing these strategies has followed. Overall modification by hydrophobic fatty acids residues or amphiphilic block copolymers represents a promising and relatively safe strategy to deliver proteins to the brain. PMID:24160902

Ion Trapping with Fast-Response Ion-Selective Microelectrodes Enhances Detection of Extracellular Ion Channel Gradients

PubMed Central

Messerli, Mark A.; Collis, Leon P.; Smith, Peter J.S.

2009-01-01

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 μm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10–55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane. PMID:19217875

Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

NASA Astrophysics Data System (ADS)

Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

2018-03-01

The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.

Plasma membrane repair and cellular damage control: the annexin survival kit.

PubMed

Draeger, Annette; Monastyrskaya, Katia; Babiychuk, Eduard B

2011-03-15

Plasmalemmal injury is a frequent event in the life of a cell. Physical disruption of the plasma membrane is common in cells that operate under conditions of mechanical stress. The permeability barrier can also be breached by chemical means: pathogens gain access to host cells by secreting pore-forming toxins and phospholipases, and the host's own immune system employs pore-forming proteins to eliminate both pathogens and the pathogen-invaded cells. In all cases, the influx of extracellular Ca(2+) is being sensed and interpreted as an "immediate danger" signal. Various Ca(2+)-dependent mechanisms are employed to enable plasma membrane repair. Extensively damaged regions of the plasma membrane can be patched with internal membranes delivered to the cell surface by exocytosis. Nucleated cells are capable of resealing their injured plasmalemma by endocytosis of the permeabilized site. Likewise, the shedding of membrane microparticles is thought to be involved in the physical elimination of pores. Membrane blebbing is a further damage-control mechanism, which is triggered after initial attempts at plasmalemmal resealing have failed. The members of the annexin protein family are ubiquitously expressed and function as intracellular Ca(2+) sensors. Most cells contain multiple annexins, which interact with distinct plasma membrane regions promoting membrane segregation, membrane fusion and--in combination with their individual Ca(2+)-sensitivity--allow spatially confined, graded responses to membrane injury. Copyright © 2011 Elsevier Inc. All rights reserved.

Improving electrokinetic microdevice stability by controlling electrolysis bubbles.

PubMed

Lee, Hwi Yong; Barber, Cedrick; Minerick, Adrienne R

2014-07-01

The voltage-operating window for many electrokinetic microdevices is limited by electrolysis gas bubbles that destabilize microfluidic system causing noise and irreproducible responses above ∼3 V DC and less than ∼1 kHz AC at 3 Vpp. Surfactant additives, SDS and Triton X-100, and an integrated semipermeable SnakeSkin® membrane were employed to control and assess electrolysis bubbles from platinum electrodes in a 180 by 70 μm, 10 mm long microchannel. Stabilized current responses at 100 V DC were observed with surfactant additives or SnakeSkin® barriers. Electrolysis bubble behaviors, visualized via video microscopy at the electrode surface and in the microchannels, were found to be influenced by surfactant function and SnakeSkin® barriers. Both SDS and Triton X-100 surfactants promoted smaller bubble diameters and faster bubble detachment from electrode surfaces via increasing gas solubility. In contrast, SnakeSkin® membranes enhanced natural convection and blocked bubbles from entering the microchannels and thus reduced current disturbances in the electric field. This data illustrated that electrode surface behaviors had substantially greater impacts on current stability than microbubbles within microchannels. Thus, physically blocking bubbles from microchannels is less effective than electrode functionalization approaches to stabilize electrokinetic microfluidic systems. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5

DOE PAGES

Huang, Li -Yun; Stuart, Christine; Takeda, Kazuyo; ...

2016-08-09

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelialmonolayer permeability with a corresponding dose- and time-dependent decrease in themore » expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Altogether, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.« less

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

PubMed Central

Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

2007-01-01

To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

In vitro studies of the blood-brain barrier using isolated brain capillaries and cultured endothelial cells.

PubMed

Goldstein, G W; Betz, A L; Bowman, P D; Dorovini-Zis, K

1986-01-01

The endothelial cells in brain capillaries are the anatomic site of the blood-brain barrier. To learn more about the biology of these specialized cells, we developed methods to prepare suspensions of purified brain microvessels as well as primary cultures of endothelial cells in monolayer. These two preparations allow for direct investigation of the metabolism, transport properties, and receptor content of the brain capillary. We used isolated brain microvessels to study distribution of membrane carriers between the luminal and the abluminal plasma membrane of endothelial cells. We found that Na+K+-ATPase and the A-system amino-acid transport system are located predominantly on the abluminal surface of brain capillary endothelial cells. This distribution of transport carriers is consistent with the low permeability of potassium and small neutral amino acids in the blood-to-brain direction. It suggests, however, that both solutes can be actively transported across brain capillaries from the brain interstitial fluid to the blood. In tissue culture, the endothelial cells form continuous tight junctions with their neighbors. This results in a cellular layer impermeable to protein tracers. When exposed to hyperosmolar solutions, in an attempt to mimic the conditions that open the blood-brain barrier in vivo, we found a reversible separation of the tight junctions between contiguous endothelial cells. No indication of activation of pinocytosis was observed. In vitro systems provide a novel approach for studying the function of the blood-brain barrier and allow for observations not possible with intact animals.

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor.

PubMed

Brown, Jacquelyn A; Pensabene, Virginia; Markov, Dmitry A; Allwardt, Vanessa; Neely, M Diana; Shi, Mingjian; Britt, Clayton M; Hoilett, Orlando S; Yang, Qing; Brewer, Bryson M; Samson, Philip C; McCawley, Lisa J; May, James M; Webb, Donna J; Li, Deyu; Bowman, Aaron B; Reiserer, Ronald S; Wikswo, John P

2015-09-01

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.

Micromachined peristaltic pump

NASA Technical Reports Server (NTRS)

Hartley, Frank T. (Inventor)

1998-01-01

A micromachined pump including a channel formed in a semiconductor substrate by conventional processes such as chemical etching. A number of insulating barriers are established in the substrate parallel to one another and transverse to the channel. The barriers separate a series of electrically conductive strips. An overlying flexible conductive membrane is applied over the channel and conductive strips with an insulating layer separating the conductive strips from the conductive membrane. Application of a sequential voltage to the series of strips pulls the membrane into the channel portion of each successive strip to achieve a pumping action. A particularly desirable arrangement employs a micromachined push-pull dual channel cavity employing two substrates with a single membrane sandwiched between them.

Micro heat barrier

DOEpatents

Marshall, Albert C.; Kravitz, Stanley H.; Tigges, Chris P.; Vawter, Gregory A.

2003-08-12

A highly effective, micron-scale micro heat barrier structure and process for manufacturing a micro heat barrier based on semiconductor and/or MEMS fabrication techniques. The micro heat barrier has an array of non-metallic, freestanding microsupports with a height less than 100 microns, attached to a substrate. An infrared reflective membrane (e.g., 1 micron gold) can be supported by the array of microsupports to provide radiation shielding. The micro heat barrier can be evacuated to eliminate gas phase heat conduction and convection. Semi-isotropic, reactive ion plasma etching can be used to create a microspike having a cusp-like shape with a sharp, pointed tip (<0.1 micron), to minimize the tip's contact area. A heat source can be placed directly on the microspikes. The micro heat barrier can have an apparent thermal conductivity in the range of 10.sup.-6 to 10.sup.-7 W/m-K. Multiple layers of reflective membranes can be used to increase thermal resistance.

Geomembrane barriers using integral fiber optics to monitor barrier integrity

DOEpatents

Staller, G.E.; Wemple, R.P.

1996-10-22

This invention provides a geomembrane or geotextile with embedded optical sensors that are used to monitor the status of containment site barriers. Fiber optic strands are used to form the sensors that can detect and monitor conditions at the sites such as breaches, slope creep, subsidence, leachate levels, fires, and types of materials present or leaking from the site. The strands are integral to the membrane or textile materials. The geosynthetic membrane is deployed at the site in a fashion similar to carpet laying. Edges of the membrane or textile are joined to form a liner and the ends of the membrane or textile become the connection zones for obtaining signals from the sensors. A connection interface with a control system to generate Optical Time Delay Response or other light signals for transmission to the optic fiber strands or sensors and also to receive reflected signals from the sensors is included in the system. Software to interpret the sensor signals can be used in the geosynthetic monitoring system. 6 figs.

Geomembrane barriers using integral fiber optics to monitor barrier integrity

DOEpatents

Staller, George E.; Wemple, Robert P.

1996-01-01

This invention provides a geomembrane or geotextile with embedded optical sensors that are used to monitor the status of containment site barriers. Fiber optic strands are used to form the sensors that can detect and monitor conditions at the sites such as breaches, slope creep, subsidence, leachate levels, fires, and types of materials present or leaking from the site. The strands are integral to the membrane or textile materials. The geosythetic membrane is deployed at the site in a fashion similar to carpet laying. Edges of the membrane or textile are joined to form a liner and the ends of the membrane or textile become the connection zones for obtaining signals from the sensors. A connection interface with a control system to generate Optical Time Delay Response or other light signals for transmission to the optic fiber strands or sensors and also to receive reflected signals from the sensors is included in the system. Software to interpret the sensor signals can be used in the geosythetic monitoring system.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation

PubMed Central

Naegeli, Kaleb M.; Chi, Qiuyi; Ziel, Joshua W.; Hagedorn, Elliott J.; Park, Jieun E.; Jayadev, Ranjay; Sherwood, David R.

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue. PMID:26765257

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

PubMed

Lohmer, Lauren L; Clay, Matthew R; Naegeli, Kaleb M; Chi, Qiuyi; Ziel, Joshua W; Hagedorn, Elliott J; Park, Jieun E; Jayadev, Ranjay; Sherwood, David R

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

[Features of the structure and parameters of the intra-alveolar septa in residents of Western Siberia].

PubMed

Shishkin, G S; Ustiuzhaninova, N V

1998-01-01

In inhabitants of Novosibirsk and Moscow district intraalveolar septum, the basic functional element of the lung consists of interstitial connective tissue and capillar network, limited with basement membranes, on which cells of epithelial lining are placed. There are no principal differences in septal structure between individuals from both regions. The majority of morphometric parameters are statistically similar, although certain peculiarities were noted in human subjects from Siberia, the most important of which is that air-blood barrier in them is 33% thinner than those in inhabitants of Moscow district. Majority of capillaries possess the air-blood barrier from both sides. This increases diffous capacity of the lungs and blood oxygenation level.

High Performance MgO-barrier Magnetic Tunnel Junctions for Flexible and Wearable Spintronic Applications

PubMed Central

Chen, Jun-Yang; Lau, Yong-Chang; Coey, J. M. D.; Li, Mo; Wang, Jian-Ping

2017-01-01

The magnetic tunnel junction (MTJ) using MgO barrier is one of most important building blocks for spintronic devices and has been widely utilized as miniaturized magentic sensors. It could play an important role in wearable medical devices if they can be fabricated on flexible substrates. The required stringent fabrication processes to obtain high quality MgO-barrier MTJs, however, limit its integration with flexible electronics devices. In this work, we have developed a method to fabricate high-performance MgO-barrier MTJs directly onto ultrathin flexible silicon membrane with a thickness of 14 μm and then transfer-and-bond to plastic substrates. Remarkably, such flexible MTJs are fully functional, exhibiting a TMR ratio as high as 190% under bending radii as small as 5 mm. The devices‘ robustness is manifested by its retained excellent performance and unaltered TMR ratio after over 1000 bending cycles. The demonstrated flexible MgO-barrier MTJs opens the door to integrating high-performance spintronic devices in flexible and wearable electronics devices for a plethora of biomedical sensing applications. PMID:28150807

High Performance MgO-barrier Magnetic Tunnel Junctions for Flexible and Wearable Spintronic Applications.

PubMed

Chen, Jun-Yang; Lau, Yong-Chang; Coey, J M D; Li, Mo; Wang, Jian-Ping

2017-02-02

The magnetic tunnel junction (MTJ) using MgO barrier is one of most important building blocks for spintronic devices and has been widely utilized as miniaturized magentic sensors. It could play an important role in wearable medical devices if they can be fabricated on flexible substrates. The required stringent fabrication processes to obtain high quality MgO-barrier MTJs, however, limit its integration with flexible electronics devices. In this work, we have developed a method to fabricate high-performance MgO-barrier MTJs directly onto ultrathin flexible silicon membrane with a thickness of 14 μm and then transfer-and-bond to plastic substrates. Remarkably, such flexible MTJs are fully functional, exhibiting a TMR ratio as high as 190% under bending radii as small as 5 mm. The devices' robustness is manifested by its retained excellent performance and unaltered TMR ratio after over 1000 bending cycles. The demonstrated flexible MgO-barrier MTJs opens the door to integrating high-performance spintronic devices in flexible and wearable electronics devices for a plethora of biomedical sensing applications.

Acid-doped polymer nanofiber framework: Three-dimensional proton conductive network for high-performance fuel cells

NASA Astrophysics Data System (ADS)

Tanaka, Manabu; Takeda, Yasushi; Wakiya, Takeru; Wakamoto, Yuta; Harigaya, Kaori; Ito, Tatsunori; Tarao, Takashi; Kawakami, Hiroyoshi

2017-02-01

High-performance polymer electrolyte membranes (PEMs) with excellent proton conductivity, gas barrier property, and membrane stability are desired for future fuel cells. Here we report the development of PEMs based on our proposed new concept "Nanofiber Framework (NfF)." The NfF composite membranes composed of phytic acid-doped polybenzimidazole nanofibers (PBINf) and Nafion matrix show higher proton conductivity than the recast-Nafion membrane without nanofibers. A series of analyses reveal the formation of three-dimensional network nanostructures to conduct protons and water effectively through acid-condensed layers at the interface of PBINf and Nafion matrix. In addition, the NfF composite membrane achieves high gas barrier property and distinguished membrane stability. The fuel cell performance by the NfF composite membrane, which enables ultra-thin membranes with their thickness less than 5 μm, is superior to that by the recast-Nafion membrane, especially at low relative humidity. Such NfF-based high-performance PEM will be accomplished not only by the Nafion matrix used in this study but also by other polymer electrolyte matrices for future PEFCs.

Oxygen-selective immobilized liquid membranes for operation of lithium-air batteries in ambient air

NASA Astrophysics Data System (ADS)

Zhang, Jian; Xu, Wu; Liu, Wei

In this work, nonaqueous electrolyte-based Li-air batteries with an O 2-selective membrane have been developed for operation in ambient air of 20-30% relative humidity (RH). The O 2 gas is continuously supplied through a membrane barrier layer at the interface of the cathode and ambient air. The membrane allows O 2 to permeate through while blocking moisture. Such membranes can be prepared by loading O 2-selective silicone oils into porous supports such as porous metal sheets and Teflon (PTFE) films. It was found that the silicone oil of high viscosity shows better performance. The immobilized silicone oil membrane in the porous PTFE film enabled the Li-air batteries with carbon black air electrodes to operate in ambient air (at 20% RH) for 16.3 days with a specific capacity of 789 mAh g -1 carbon and a specific energy of 2182 Wh kg -1 carbon. Its performance is much better than a reference battery assembled with a commercial, porous PTFE diffusion membranes as the moisture barrier layer on the cathode, which only had a discharge time of 5.5 days corresponding to a specific capacity of 267 mAh g -1 carbon and a specific energy of 704 Wh kg -1 carbon. The Li-air battery with the present selective membrane barrier layer even showed better performance in ambient air operation (20% RH) than the reference battery tested in the dry air box (<1% RH).

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

PubMed Central

Simpson, Brent W.; Owens, Tristan W.; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine M.; Trauger, Sunia A.

2016-01-01

ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. PMID:27795402

A facile and efficient approach for pore-opening detection of anodic aluminum oxide membranes

NASA Astrophysics Data System (ADS)

Cui, Jiewu; Wu, Yucheng; Wang, Yan; Zheng, Hongmei; Xu, Guangqing; Zhang, Xinyi

2012-05-01

The well aligned porous anodic aluminum oxide (AAO) membrane is fabricated by a two-step anodization method. The oxide barrier layer of AAO membrane must be removed to get through-hole membrane for synthesizing nanowires and nanotubes of metals, semiconductors and conducting polymers. Removal of the barrier layer of oxide and pore-extending is of significant importance for the preparation of AAO membrane with through-hole pore morphology and desired pore diameter. The conventional method for pore opening is that AAO membrane after removing of aluminum substrate is immersed in chemical etching solution, which is completely empirical and results in catastrophic damage for AAO membrane frequently. A very simple and efficient approach based on capillary action for detecting pore opening of AAO membrane is introduced in this paper, this method can achieve the detection for pore opening visually and control the pore diameter precisely to get desired morphology and the pore diameter of AAO membrane. Two kinds of AAO membranes with different pore shape were obtained by different pore opening methods. In addition, one-dimensional gradient gold nanowires are also fabricated by electrodeposition based on AAO membranes.

Spectrin-ankyrin interaction mechanics: A key force balance factor in the red blood cell membrane skeleton.

PubMed

Saito, Masakazu; Watanabe-Nakayama, Takahiro; Machida, Shinichi; Osada, Toshiya; Afrin, Rehana; Ikai, Atsushi

2015-01-01

As major components of red blood cell (RBC) cytoskeleton, spectrin and F-actin form a network that covers the entire cytoplasmic surface of the plasma membrane. The cross-linked two layered structure, called the membrane skeleton, keeps the structural integrity of RBC under drastically changing mechanical environment during circulation. We performed force spectroscopy experiments on the atomic force microscope (AFM) as a means to clarify the mechanical characteristics of spectrin-ankyrin interaction, a key factor in the force balance of the RBC cytoskeletal structure. An AFM tip was functionalized with ANK1-62k and used to probe spectrin crosslinked to mica surface. A force spectroscopy study gave a mean unbinding force of ~30 pN under our experimental conditions. Two energy barriers were identified in the unbinding process. The result was related to the well-known flexibility of spectrin tetramer and participation of ankyrin 1-spectrin interaction in the overall balance of membrane skeleton dynamics. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of chain rigidity on the conformation of model lipid membranes in the presence of cylindrical nanoparticle inclusions

NASA Astrophysics Data System (ADS)

Diloreto, Chris; Wickham, Robert

2012-02-01

We employ real-space self-consistent field theory to study the conformation of model lipid membranes in the presence of solvent and cylindrical nanoparticle inclusions (''peptides''). Whereas it is common to employ a polymeric Gaussian chain model for the lipids, here we model the lipids as persistent, worm-like chains. Our motivation is to develop a more realistic field theory to describe the action of pore-forming anti-microbial peptides that disrupt the bacterial cell membrane. We employ operator-splitting and a pseudo-spectral algorithm, using SpharmonicKit for the chain tangent degrees of freedom, to solve for the worm-like chain propagator. The peptides, modelled using a mask function, have a surface patterned with hydrophobic and hydrophillic patches, but no charge. We examine the role chain rigidity plays in the hydrophobic mismatch, the membrane-mediated interaction between two peptides, the size and structure of pores formed by peptide aggregates, and the free-energy barrier for peptide insertion into the membrane. Our results suggest that chain rigidity influences both the pore structure and the mechanism of pore formation.

Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota

PubMed Central

Cian, Raúl E.; Drago, Silvina R.; Sánchez de Medina, Fermín; Martínez-Augustin, Olga

2015-01-01

Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain “cellulose binding domains”, phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and “dl-hybrid”) and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function. PMID:26308006

Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota.

PubMed

Cian, Raúl E; Drago, Silvina R; de Medina, Fermín Sánchez; Martínez-Augustin, Olga

2015-08-20

Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain "cellulose binding domains", phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and "dl-hybrid") and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function.

Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

PubMed

Szabo, Roman; Peters, Diane E; Kosa, Peter; Camerer, Eric; Bugge, Thomas H

2014-07-01

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.

Regulation of Feto-Maternal Barrier by Matriptase- and PAR-2-Mediated Signaling Is Required for Placental Morphogenesis and Mouse Embryonic Survival

PubMed Central

Szabo, Roman; Peters, Diane E.; Kosa, Peter; Camerer, Eric; Bugge, Thomas H.

2014-01-01

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival. PMID:25078604

Separating attoliter-sized compartments using fluid pore-spanning lipid bilayers.

PubMed

Lazzara, Thomas D; Carnarius, Christian; Kocun, Marta; Janshoff, Andreas; Steinem, Claudia

2011-09-27

Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55-60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5-15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging. © 2011 American Chemical Society

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase.

PubMed

Villalba-Galea, Carlos A; Frezza, Ludivine; Sandtner, Walter; Bezanilla, Francisco

2013-11-01

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.

Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay.

PubMed

Okamoto, K; Gotoh, N; Nishino, T

2001-07-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC beta-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC beta-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC beta-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC beta-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC beta-lactamase but not from the lower affinity of PBPs for penems.

Pseudomonas aeruginosa Reveals High Intrinsic Resistance to Penem Antibiotics: Penem Resistance Mechanisms and Their Interplay

PubMed Central

Okamoto, Kiyomi; Gotoh, Naomasa; Nishino, Takeshi

2001-01-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC β-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC β-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC β-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC β-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC β-lactamase but not from the lower affinity of PBPs for penems. PMID:11408209

The effects of polar excipients transcutol and dexpanthenol on molecular mobility, permeability, and electrical impedance of the skin barrier.

PubMed

Björklund, Sebastian; Pham, Quoc Dat; Jensen, Louise Bastholm; Knudsen, Nina Østergaard; Nielsen, Lars Dencker; Ekelund, Katarina; Ruzgas, Tautgirdas; Engblom, Johan; Sparr, Emma

2016-10-01

In the development of transdermal and topical products it is important to understand how formulation ingredients interact with the molecular components of the upper layer of the skin, the stratum corneum (SC), and thereby influence its macroscopic barrier properties. The aim here was to investigate the effect of two commonly used excipients, transcutol and dexpanthenol, on the molecular as well as the macroscopic properties of the skin membrane. Polarization transfer solid-state NMR methods were combined with steady-state flux and impedance spectroscopy measurements to investigate how these common excipients influence the molecular components of SC and its barrier function at strictly controlled hydration conditions in vitro with excised porcine skin. The NMR results provide completely new molecular insight into how transcutol and dexpanthenol affect specific molecular segments of both SC lipids and proteins. The presence of transcutol or dexpanthenol in the formulation at fixed water activity results in increased effective skin permeability of the model drug metronidazole. Finally, impedance spectroscopy data show clear changes of the effective skin capacitance after treatment with transcutol or dexpanthenol. Based on the complementary data, we are able to draw direct links between effects on the molecular properties and on the macroscopic barrier function of the skin barrier under treatment with formulations containing transcutol or dexpanthenol. Copyright © 2016 Elsevier Inc. All rights reserved.

A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

PubMed

Mollet, Björne B; Bogaerts, Iven L J; van Almen, Geert C; Dankers, Patricia Y W

2017-06-01

Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Penetration of alkali atoms throughout a graphene membrane: theoretical modeling

NASA Astrophysics Data System (ADS)

Boukhvalov, D. W.; Virojanadara, C.

2012-02-01

Theoretical studies of penetration of various alkali atoms (Li, Na, Rb, Cs) throughout a graphene membrane grown on a silicon carbide substrate are reported and compared with recent experimental results. Results of first principles modeling demonstrate a rather low (about 0.8 eV) energy barrier for the formation of temporary defects in the carbon layer required for the penetration of Li at a high concentration of adatoms, a higher (about 2 eV) barrier for Na, and barriers above 4 eV for Rb and Cs. Experiments prove migration of lithium adatoms from the graphene surface to the buffer layer and SiC substrate at room temperature, sodium at 100 °C and impenetrability of the graphene membrane for Rb and Cs. Differences between epitaxial and free-standing graphene for the penetration of alkali ions are also discussed.

Penetration of alkali atoms throughout a graphene membrane: theoretical modeling.

PubMed

Boukhvalov, D W; Virojanadara, C

2012-03-07

Theoretical studies of penetration of various alkali atoms (Li, Na, Rb, Cs) throughout a graphene membrane grown on a silicon carbide substrate are reported and compared with recent experimental results. Results of first principles modeling demonstrate a rather low (about 0.8 eV) energy barrier for the formation of temporary defects in the carbon layer required for the penetration of Li at a high concentration of adatoms, a higher (about 2 eV) barrier for Na, and barriers above 4 eV for Rb and Cs. Experiments prove migration of lithium adatoms from the graphene surface to the buffer layer and SiC substrate at room temperature, sodium at 100 °C and impenetrability of the graphene membrane for Rb and Cs. Differences between epitaxial and free-standing graphene for the penetration of alkali ions are also discussed.

Importance of balancing membrane and electrode water in anion exchange membrane fuel cells

NASA Astrophysics Data System (ADS)

Omasta, T. J.; Wang, L.; Peng, X.; Lewis, C. A.; Varcoe, J. R.; Mustain, W. E.

2018-01-01

Anion exchange membrane fuel cells (AEMFCs) offer several potential advantages over proton exchange membrane fuel cells (PEMFCs), most notably to overcome the cost barrier that has slowed the growth and large scale implementation of fuel cells for transportation. However, limitations in performance have held back AEMFCs, specifically in the areas of stability, carbonation, and maximum achievable current and power densities. In order for AEMFCs to contend with PEMFCs for market viability, it is necessary to realize a competitive cell performance. This work demonstrates a new benchmark for a H2/O2 AEMFC with a peak power density of 1.4 W cm-2 at 60 °C. This was accomplished by taking a more precise look at balancing necessary membrane hydration while preventing electrode flooding, which somewhat surprisingly can occur both at the anode and the cathode. Specifically, radiation-grafted ETFE-based anion exchange membranes and anion exchange ionomer powder, functionalized with benchmark benzyltrimethylammonium groups, were utilized to examine the effects of the following parameters on AEMFC performance: feed gas flow rate, the use of hydrophobic vs. hydrophilic gas diffusion layers, and gas feed dew points.

Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

PubMed

Lau, Julia B; Stork, Simone; Moog, Daniel; Schulz, Julian; Maier, Uwe G

2016-04-01

Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein-protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD-related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids. © 2015 John Wiley & Sons Ltd.

Selective Antimicrobial Activities and Action Mechanism of Micelles Self-Assembled by Cationic Oligomeric Surfactants.

PubMed

Zhou, Chengcheng; Wang, Fengyan; Chen, Hui; Li, Meng; Qiao, Fulin; Liu, Zhang; Hou, Yanbo; Wu, Chunxian; Fan, Yaxun; Liu, Libing; Wang, Shu; Wang, Yilin

2016-02-17

This work reports that cationic micelles formed by cationic trimeric, tetrameric, and hexameric surfactants bearing amide moieties in spacers can efficiently kill Gram-negative E. coli with a very low minimum inhibitory concentration (1.70-0.93 μM), and do not cause obvious toxicity to mammalian cells at the concentrations used. With the increase of the oligomerization degree, the antibacterial activity of the oligomeric surfactants increases, i.e., hexameric surfactant > tetrameric surfactant > trimeric surfactant. Isothermal titration microcalorimetry, scanning electron microscopy, and zeta potential results reveal that the cationic micelles interact with the cell membrane of E. coli through two processes. First, the integrity of outer membrane of E. coli is disrupted by the electrostatic interaction of the cationic ammonium groups of the surfactants with anionic groups of E. coli, resulting in loss of the barrier function of the outer membrane. The inner membrane then is disintegrated by the hydrophobic interaction of the surfactant hydrocarbon chains with the hydrophobic domains of the inner membrane, leading to the cytoplast leakage. The formation of micelles of these cationic oligomeric surfactants at very low concentration enables more efficient interaction with bacterial cell membrane, which endows the oligomeric surfactants with high antibacterial activity.

Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

PubMed Central

Walsh, Dustin R.; Nolin, Thomas D.

2015-01-01

Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

Brazing method

DOEpatents

McCormick, James T.; Ferry, Paul B.; Hall, John C.

1981-10-06

There is disclosed a positive cathode electrode structure formed by brazing a thin porous membrane to a backing material by preselecting a predetermined area of the thin porous membrane and thereafter providing a braze flow barrier throughout the remainder of the membrane and electrolessly plating a nickel-phosphide alloy on the backing material, or in this case the honeycomb structure. The preselected area of the thin porous membrane is placed in intimate contact with the electrolessly plated portion of the backing material and heated to elevated temperatures in the absence of oxygen to form a brazed joint limited to a preselected area. If the braze flow barrier is provided by application of a liquid organic solvent, then the organic solvent is driven off by maintaining the thin porous membrane at elevated temperatures for an extended period of time prior to the brazing operation.

Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halwachs, Sandra

In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim,more » chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B > A) directed transport of [{sup 14}C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates. - Highlights: • Generation of MDCKII-rbABCG2 monolayers with epithelial barrier function • Detection of rbABCG2 in the apical plasma membrane of polarized MDCKII cells • Several pesticides interact with the ABCG2 transporter from rabbit placenta. • rbABCG2 mediates transport of the insecticide chlorpyrifos. • MDCKII-rbABCG2 cells are a suitable model to study transport in rabbit placenta.« less

Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro

PubMed Central

Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

2014-01-01

Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-l-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. PMID:25339691

Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+ Escherichia coli on intestinal cells.

PubMed

Roselli, Marianna; Finamore, Alberto; Hynönen, Ulla; Palva, Airi; Mengheri, Elena

2016-09-29

The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698 T is able to reduce the enterotoxigenic F4 + Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698 T protects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698 T cell wall components in the protection against F4 + ETEC infection using the intestinal Caco-2 cell line. Purified cell wall fragments (CWF) from L. amylovorus DSM 16698 T were used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4 + ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4 + ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4 + ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot. We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4 + ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4 + ETEC, inhibited membrane damage and prevented NF-kB activation. The results indicate that the various components of L. amylovorus DSM 16698 T cell wall may counteract the damage caused by F4 + ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4 + ETEC infection. U-CWF are not able to defend the cells when they are infected with F4 + ETEC but may activate protective mechanisms before pathogen infection.

Using the Drosophila Nephrocyte to Model Podocyte Function and Disease

PubMed Central

Helmstädter, Martin; Huber, Tobias B.; Hermle, Tobias

2017-01-01

Glomerular disorders are a major cause of end-stage renal disease and effective therapies are often lacking. Nephrocytes are considered to be part of the Drosophila excretory system and form slit diaphragms across cellular membrane invaginations. Nehphrocytes have been shown to share functional, morphological, and molecular features with podocytes, which form the glomerular filter in vertebrates. Here, we report the progress and the evolving tool-set of this model system. Combining a functional, accessible slit diaphragm with the power of the genetic tool-kit in Drosophila, the nephrocyte has the potential to greatly advance our understanding of the glomerular filtration barrier in health and disease. PMID:29270398

Phosphatidylcholine Membrane Fusion Is pH-Dependent.

PubMed

Akimov, Sergey A; Polynkin, Michael A; Jiménez-Munguía, Irene; Pavlov, Konstantin V; Batishchev, Oleg V

2018-05-03

Membrane fusion mediates multiple vital processes in cell life. Specialized proteins mediate the fusion process, and a substantial part of their energy is used for topological rearrangement of the membrane lipid matrix. Therefore, the elastic parameters of lipid bilayers are of crucial importance for fusion processes and for determination of the energy barriers that have to be crossed for the process to take place. In the case of fusion of enveloped viruses (e.g., influenza) with endosomal membrane, the interacting membranes are in an acidic environment, which can affect the membrane's mechanical properties. This factor is often neglected in the analysis of virus-induced membrane fusion. In the present work, we demonstrate that even for membranes composed of zwitterionic lipids, changes of the environmental pH in the physiologically relevant range of 4.0 to 7.5 can affect the rate of the membrane fusion notably. Using a continual model, we demonstrated that the key factor defining the height of the energy barrier is the spontaneous curvature of the lipid monolayer. Changes of this parameter are likely to be caused by rearrangements of the polar part of lipid molecules in response to changes of the pH of the aqueous solution bathing the membrane.

The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

PubMed Central

Grafmüller, Andrea; Shillcock, Julian; Lipowsky, Reinhard

2009-01-01

The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail inserted in each membrane. To determine the corresponding energy barrier, we measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall, three subprocesses have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8–15 kBT. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before the tensionless membrane state is attained. This would imply that the tension has to exceed a certain threshold value to induce fusion. PMID:19348749

Advances in Meningeal Immunity.

PubMed

Rua, Rejane; McGavern, Dorian B

2018-06-01

The central nervous system (CNS) is an immunologically specialized tissue protected by a blood-brain barrier. The CNS parenchyma is enveloped by a series of overlapping membranes that are collectively referred to as the meninges. The meninges provide an additional CNS barrier, harbor a diverse array of resident immune cells, and serve as a crucial interface with the periphery. Recent studies have significantly advanced our understanding of meningeal immunity, demonstrating how a complex immune landscape influences CNS functions under steady-state and inflammatory conditions. The location and activation state of meningeal immune cells can profoundly influence CNS homeostasis and contribute to neurological disorders, but these cells are also well equipped to protect the CNS from pathogens. In this review, we discuss advances in our understanding of the meningeal immune repertoire and provide insights into how this CNS barrier operates immunologically under conditions ranging from neurocognition to inflammatory diseases. Published by Elsevier Ltd.

Regulation of the basement membrane by epithelia generated forces

NASA Astrophysics Data System (ADS)

Tanner, Kandice

2012-12-01

Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre-invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor.

The Role of Sphingolipids on Innate Immunity to Intestinal Salmonella Infection.

PubMed

Huang, Fu-Chen

2017-08-07

Salmonella spp. remains a major public health problem for the whole world. To reduce the use of antimicrobial agents and drug-resistant Salmonella , a better strategy is to explore alternative therapy rather than to discover another antibiotic. Sphingolipid- and cholesterol-enriched lipid microdomains attract signaling proteins and orchestrate them toward cell signaling and membrane trafficking pathways. Recent studies have highlighted the crucial role of sphingolipids in the innate immunity against infecting pathogens. It is therefore mandatory to exploit the role of the membrane sphingolipids in the innate immunity of intestinal epithelia infected by this pathogen. In the present review, we focus on the role of sphingolipids in the innate immunity of intestinal epithelia against Salmonella infection, including adhesion, autophagy, bactericidal effect, barrier function, membrane trafficking, cytokine and antimicrobial peptide expression. The intervention of sphingolipid-enhanced foods to make our life healthy or pharmacological agents regulating sphingolipids is provided at the end.

The minimalist architectures of viroporins and their therapeutic implications

PubMed Central

OuYang, Bo; Chou, James J.

2014-01-01

Many viral genomes encode small, integral membrane proteins that form homo-oligomeric channels in membrane, and they transport protons, cations, and other molecules across the membrane barrier to aid various steps of viral entry and maturation. These viral proteins, collectively named viroporins, are crucial for viral pathogenicity. In the past five years, structures obtained by nuclear magnetic resonance (NMR), X-ray crystallography, and electron microscopy (EM) showed that viroporins often adopt minimalist architectures to achieve their functions. A number of small molecules have been identified to interfere with their channel activity and thereby inhibit viral infection, making viroporins potential drug targets for therapeutic intervention. The known architectures and inhibition mechanisms of viroporins differ significantly from each other, but some common principles are shared between them. This review article summarizes the recent developments in the structural investigation of viroporins and their inhibition by antiviral compounds. PMID:24055819

Membrane pore formation in atomistic and coarse-grained simulations.

PubMed

Kirsch, Sonja A; Böckmann, Rainer A

2016-10-01

Biological cells and their organelles are protected by ultra thin membranes. These membranes accomplish a broad variety of important tasks like separating the cell content from the outer environment, they are the site for cell-cell interactions and many enzymatic reactions, and control the in- and efflux of metabolites. For certain physiological functions e.g. in the fusion of membranes and also in a number of biotechnological applications like gene transfection the membrane integrity needs to be compromised to allow for instance for the exchange of polar molecules across the membrane barrier. Mechanisms enabling the transport of molecules across the membrane involve membrane proteins that form specific pores or act as transporters, but also so-called lipid pores induced by external fields, stress, or peptides. Recent progress in the simulation field enabled to closely mimic pore formation as supposed to occur in vivo or in vitro. Here, we review different simulation-based approaches in the study of membrane pores with a focus on lipid pore properties such as their size and energetics, poration mechanisms based on the application of external fields, charge imbalances, or surface tension, and on pores that are induced by small molecules, peptides, and lipids. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. Copyright © 2015 Elsevier B.V. All rights reserved.

Connective tissue graft as a biological barrier for guided tissue regeneration in intrabony defects: a histological study in dogs.

PubMed

Ribeiro, Fernando Salimon; Pontes, Ana Emília Farias; Zuza, Elizangela Partata; da Silva, Vanessa Camila; Lia, Raphael Carlos Comelli; Marcantonio Junior, Elcio

2015-06-01

The use of the autogenous periosteal graft as biological barrier has been proposed for periodontal regeneration. The aim of this study was to evaluate the histometric findings of the subepithelial connective tissue graft as barrier in intrabony defects compared to a bioabsorbable membrane. Three-walled intrabony defects were created surgically in the mesial aspect of the right and left maxillary canines in five healthy mongrel dogs. The defects were chronified, and two types of barriers were randomly carried out for guided tissue regeneration in a split-mouth design: the test group with a subepithelial connective tissue graft and the control group with a bioabsorbable membrane. The specimens were processed for histometric analyses of the epithelium (E), connective tissue (CT), newly formed cementum (NC), new bone (NB), and total newly formed tissues (NFT). The test side showed smaller mean of NC (3.6 ± 1.2), NB (2.1 ± 0.7), and NFT (7.7 ± 0.8) than the control group (NC 7.3 ± 0.5; NB 5.3 ± 1.3; NFT 10.1 ± 2.2; P < 0.05). No statistically significant differences were verified for E (test 3.1 ± 2.0; control 2.8 ± 2.1; P > 0.05) and CT (test 2.5 ± 1.1; control 2.0 ± 0.5; P > 0.05) between groups. The bioabsorbable membrane was more effective in maintaining the space for periodontal regeneration than periosteal connective graft when used as barrier. The bioabsorbable membrane showed more favorable regenerative results in intrabony defects in dogs than the subepithelial connective tissue graft as biological barrier.

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli

PubMed Central

Ruiz, Natividad; Gronenberg, Luisa S.; Kahne, Daniel; Silhavy, Thomas J.

2008-01-01

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM. PMID:18375759

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli.

PubMed

Ruiz, Natividad; Gronenberg, Luisa S; Kahne, Daniel; Silhavy, Thomas J

2008-04-08

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM.

Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling1[OPEN

PubMed Central

2016-01-01

Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling.

PubMed

Noctor, Graham; Foyer, Christine H

2016-07-01

Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. © 2016 American Society of Plant Biologists. All Rights Reserved.

The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation.

PubMed

Kawase, Tomoyuki; Kamiya, Mana; Kobayashi, Mito; Tanaka, Takaaki; Okuda, Kazuhiro; Wolff, Larry F; Yoshie, Hiromasa

2015-05-01

Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment. © 2014 Wiley Periodicals, Inc.

Intraepithelial gammadelta+ lymphocytes maintain the integrity of intestinal epithelial tight junctions in response to infection.

PubMed

Dalton, Jane E; Cruickshank, Sheena M; Egan, Charlotte E; Mears, Rainy; Newton, Darren J; Andrew, Elizabeth M; Lawrence, Beth; Howell, Gareth; Else, Kathryn J; Gubbels, Marc-Jan; Striepen, Boris; Smith, Judith E; White, Stanley J; Carding, Simon R

2006-09-01

Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.

Methods for fabricating a micro heat barrier

DOEpatents

Marshall, Albert C.; Kravitz, Stanley H.; Tigges, Chris P.; Vawter, Gregory A.

2004-01-06

Methods for fabricating a highly effective, micron-scale micro heat barrier structure and process for manufacturing a micro heat barrier based on semiconductor and/or MEMS fabrication techniques. The micro heat barrier has an array of non-metallic, freestanding microsupports with a height less than 100 microns, attached to a substrate. An infrared reflective membrane (e.g., 1 micron gold) can be supported by the array of microsupports to provide radiation shielding. The micro heat barrier can be evacuated to eliminate gas phase heat conduction and convection. Semi-isotropic, reactive ion plasma etching can be used to create a microspike having a cusp-like shape with a sharp, pointed tip (<0.1 micron), to minimize the tip's contact area. A heat source can be placed directly on the microspikes. The micro heat barrier can have an apparent thermal conductivity in the range of 10.sup.-6 to 10.sup.-7 W/m-K. Multiple layers of reflective membranes can be used to increase thermal resistance.

Inner Blood-Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig.

PubMed

Zhang, Zhengyu; Uchida, Yasuo; Hirano, Satoshi; Ando, Daisuke; Kubo, Yoshiyuki; Auriola, Seppo; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi; Urtti, Arto; Terasaki, Tetsuya; Tachikawa, Masanori

2017-11-06

The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood-retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood-brain barrier (BBB), and blood-cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/μg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/μg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/μg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/μg protein), and sodium-potassium adenosine triphosphatase (Na + /K + -ATPase; 53.7 fmol/μg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB (R 2 = 0.8906), followed by outer BRB (R 2 = 0.7988) and BCSFB (R 2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6) and multidrug resistance-associated protein 1 (MRP1, ABCC1) were expressed at the outer BRB (0.378 and 1.03 fmol/μg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.

Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development*

PubMed Central

Wong, Bernice H.; Chan, Jia Pei; Cazenave-Gassiot, Amaury; Poh, Rebecca W.; Foo, Juat Chin; Galam, Dwight L. A.; Ghosh, Sujoy; Nguyen, Long N.; Barathi, Veluchamy A.; Yeo, Sia W.; Luu, Chi D.; Wenk, Markus R.; Silver, David L.

2016-01-01

Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo. Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs. PMID:27008858

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

PubMed

Uhl, P B; Szober, C M; Amann, B; Alge-Priglinger, C; Ueffing, M; Hauck, S M; Deeg, C A

2014-09-23

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein kinase C restricts transport of carnitine by amino acid transporter ATB(0,+) apically localized in the blood-brain barrier.

PubMed

Michalec, Katarzyna; Mysiorek, Caroline; Kuntz, Mélanie; Bérézowski, Vincent; Szczepankiewicz, Andrzej A; Wilczyński, Grzegorz M; Cecchelli, Roméo; Nałęcz, Katarzyna A

2014-07-15

Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated. Copyright © 2014 Elsevier Inc. All rights reserved.

Bilateral PLA/alginate membranes for the prevention of postsurgical adhesions.

PubMed

Kessler, Martina; Esser, Eva; Groll, Jürgen; Tessmar, Jörg

2016-11-01

A bilateral barrier membrane for the prevention of postsurgical adhesions was developed. Thereby, a smooth PLA side was supposed to keep the affected tissues glidingly separated, while a mucoadhesive side made of alginate was meant to keep the barrier resident on the site of injury so that suturing becomes redundant or at least the membrane stays long enough to facilitate surgical handling. Because hydrophilic alginate and lipophilic PLA films show only low cohesion, solution electrospun meshes of PLA and PLA-PEG-PLA triblock copolymers with varying poly(ethylene glycol) [PEG] content were investigated as cohesion promoter to avoid an easy separation of the functionally different layers. Using direct electrospinning onto the PLA film, a modified contact surface of the mesh was created, which allowed the tested alginate solutions (3%, 5%) to infiltrate to different extents. Thereby, an increasing content of hydrophilic PEG within the mesh copolymer and a lower alginate concentration facilitated the infiltration. As a result, the PLA film with a PLA35k-PEG10k-PLA35k (racemic PLA chains) mesh and an alginate layer cast from a 3% alginate solution appeared to be the most effective combination as examined by means of a t peel test, a mucoadhesion test, a tensile test and optical evaluations. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1563-1570, 2016. © 2015 Wiley Periodicals, Inc.

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE PAGES

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

2015-01-02

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Effect of vilon and epithalon on activity of enzymes in epithelial and subepithelial layers in small intestine of old rats.

PubMed

Khavinson, V Kh; Timofeeva, N M; Malinin, V V; Gordova, L A; Nikitina, A A

2002-12-01

Per os administration of Vilon (Lys-Glu) or Epithalon (Ala-Glu-Asp-Gly) to aged Wistar rats for 1 month significantly increased activity of membrane enzymes maltase and alkaline phosphatase in epithelial layer of the small intestine. In addition, Vilon significantly increased activity of cytosolic glycyl-L-leucine dipeptidase in the stromal and seromuscular layers of the small intestine in comparison with the control rats not treated with this agent. These findings suggest improvement of trophic and barrier functions of the small intestine and corroborate the hypothesis on the existence of not only epithelial, but also subepithelial enzymatic barrier supporting the enzyme system in the small intestine, especially in aged animals.

Phospholipase D function in Saccharomyces cerevisiae.

PubMed

Mendonsa, Rima; Engebrecht, JoAnne

2009-09-01

Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.

Water Permeation Across Biological Membranes: Mechanism and Dynamics of Aquaporin-1 and GlpF

NASA Astrophysics Data System (ADS)

de Groot, Bert L.; Grubmüller, Helmut

2001-12-01

``Real time'' molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented. We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels. Both proteins act as two-stage filters: Conserved fingerprint [asparagine-proline-alanine (NPA)] motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter. Hydrophobic regions near the NPA motifs are rate-limiting water barriers. In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity. In GlpF, a glycerol-mediated ``induced fit'' gating motion is proposed to generate selectivity for glycerol over water.

Caveolins and caveolae in ocular physiology and pathophysiology.

PubMed

Gu, Xiaowu; Reagan, Alaina M; McClellan, Mark E; Elliott, Michael H

2017-01-01

Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., "lipid rafts") have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signaling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function. Copyright © 2016 Elsevier Ltd. All rights reserved.

Caveolins and caveolae in ocular physiology and pathophysiology

PubMed Central

Gu, Xiaowu; Reagan, Alaina M.; McClellan, Mark E.; Elliott, Michael H.

2016-01-01

Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., “lipid rafts”) have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signalling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function. PMID:27664379

Accelerated molecular dynamics and protein conformational change: a theoretical and practical guide using a membrane embedded model neurotransmitter transporter.

PubMed

Gedeon, Patrick C; Thomas, James R; Madura, Jeffry D

2015-01-01

Molecular dynamics simulation provides a powerful and accurate method to model protein conformational change, yet timescale limitations often prevent direct assessment of the kinetic properties of interest. A large number of molecular dynamic steps are necessary for rare events to occur, which allow a system to overcome energy barriers and conformationally transition from one potential energy minimum to another. For many proteins, the energy landscape is further complicated by a multitude of potential energy wells, each separated by high free-energy barriers and each potentially representative of a functionally important protein conformation. To overcome these obstacles, accelerated molecular dynamics utilizes a robust bias potential function to simulate the transition between different potential energy minima. This straightforward approach more efficiently samples conformational space in comparison to classical molecular dynamics simulation, does not require advanced knowledge of the potential energy landscape and converges to the proper canonical distribution. Here, we review the theory behind accelerated molecular dynamics and discuss the approach in the context of modeling protein conformational change. As a practical example, we provide a detailed, step-by-step explanation of how to perform an accelerated molecular dynamics simulation using a model neurotransmitter transporter embedded in a lipid cell membrane. Changes in protein conformation of relevance to the substrate transport cycle are then examined using principle component analysis.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

PubMed

Vacca, Ophélie; Charles-Messance, Hugo; El Mathari, Brahim; Sene, Abdoulaye; Barbe, Peggy; Fouquet, Stéphane; Aragón, Jorge; Darche, Marie; Giocanti-Aurégan, Audrey; Paques, Michel; Sahel, José-Alain; Tadayoni, Ramin; Montañez, Cecilia; Dalkara, Deniz; Rendon, Alvaro

2016-07-15

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR). © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Thermodynamics of Micelle Formation and Membrane Fusion Modulate Antimicrobial Lipopeptide Activity

PubMed Central

Lin, Dejun; Grossfield, Alan

2015-01-01

Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs’ ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads. PMID:26287627

Thin-film Nanofibrous Composite Membranes Containing Cellulose or Chitin Barrier Layers Fabricated by Ionic Liquids

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Ma; B Hsiao; B Chu

The barrier layer of high-flux ultrafiltration (UF) thin-film nanofibrous composite (TFNC) membranes for purification of wastewater (e.g., bilge water) have been prepared by using cellulose, chitin, and a cellulose-chitin blend, regenerated from an ionic liquid. The structures and properties of regenerated cellulose, chitin, and a cellulose-chitin blend were analyzed with thermogravimetric analysis (TGA) and wide-angle X-ray diffraction (WAXD). The surface morphology, pore size and pore size distribution of TFNC membranes were determined by SEM images and molecular weight cut-off (MWCO) methods. An oil/water emulsion, a model of bilge water, was used as the feed solution, and the permeation flux andmore » rejection ratio of the membranes were investigated. TFNC membranes based on the cellulose-chitin blend exhibited 10 times higher permeation flux when compared with a commercial UF membrane (PAN10, Sepro) with a similar rejection ratio after filtration over a time period of up to 100 h, implying the practical feasibility of such membranes for UF applications.« less

Bias-Exchange Metadynamics Simulation of Membrane Permeation of 20 Amino Acids.

PubMed

Cao, Zanxia; Bian, Yunqiang; Hu, Guodong; Zhao, Liling; Kong, Zhenzhen; Yang, Yuedong; Wang, Jihua; Zhou, Yaoqi

2018-03-16

Thermodynamics of the permeation of amino acids from water to lipid bilayers is an important first step for understanding the mechanism of cell-permeating peptides and the thermodynamics of membrane protein structure and stability. In this work, we employed bias-exchange metadynamics simulations to simulate the membrane permeation of all 20 amino acids from water to the center of a dipalmitoylphosphatidylcholine (DPPC) membrane (consists of 256 lipids) by using both directional and torsion angles for conformational sampling. The overall accuracy for the free energy profiles obtained is supported by significant correlation coefficients (correlation coefficient at 0.5-0.6) between our results and previous experimental or computational studies. The free energy profiles indicated that (1) polar amino acids have larger free energy barriers than nonpolar amino acids; (2) negatively charged amino acids are the most difficult to enter into the membrane; and (3) conformational transitions for many amino acids during membrane crossing is the key for reduced free energy barriers. These results represent the first set of simulated free energy profiles of membrane crossing for all 20 amino acids.

3D-printed membrane for guided tissue regeneration.

PubMed

Tayebi, Lobat; Rasoulianboroujeni, Morteza; Moharamzadeh, Keyvan; Almela, Thafar K D; Cui, Zhanfeng; Ye, Hua

2018-03-01

Three-dimensional (3D) printing is currently being intensely studied for a diverse set of applications, including the development of bioengineered tissues, as well as the production of functional biomedical materials and devices for dental and orthopedic applications. The aim of this study was to develop and characterize a 3D-printed hybrid construct that can be potentially suitable for guided tissue regeneration (GTR). For this purpose, the rheology analyses have been performed on different bioinks and a specific solution comprising 8% gelatin, 2% elastin and 0.5% sodium hyaluronate has been selected as the most suitable composition for printing a structured membrane for GTR application. Each membrane is composed of 6 layers with strand angles from the first layer to the last layer of 45, 135, 0, 90, 0 and 90°. Confirmed by 3D Laser Measuring imaging, the membrane has small pores on one side and large pores on the other to be able to accommodate different cells like osteoblasts, fibroblasts and keratinocytes on different sides. The ultimate cross-linked product is a 150μm thick flexible and bendable membrane with easy surgical handling. Static and dynamic mechanical testing revealed static tensile modules of 1.95±0.55MPa and a dynamic tensile storage modulus of 314±50kPa. Through seeding the membranes with fibroblast and keratinocyte cells, the results of in vitro tests, including histological analysis, tissue viability examinations and DAPI staining, indicated that the membrane has desirable in vitro biocompatibility. The membrane has demonstrated the barrier function of a GTR membrane by thorough separation of the oral epithelial layer from the underlying tissues. In conclusion, we have characterized a biocompatible and bio-resorbable 3D-printed structured gelatin/elastin/sodium hyaluronate membrane with optimal biostability, mechanical strength and surgical handling characteristics in terms of suturability for potential application in GTR procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiple Roles of Integrin-Linked Kinase in Epidermal Development, Maturation and Pigmentation Revealed by Molecular Profiling

PubMed Central

Judah, David; Rudkouskaya, Alena; Wilson, Ryan; Carter, David E.; Dagnino, Lina

2012-01-01

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function. PMID:22574216

Multiple roles of integrin-linked kinase in epidermal development, maturation and pigmentation revealed by molecular profiling.

PubMed

Judah, David; Rudkouskaya, Alena; Wilson, Ryan; Carter, David E; Dagnino, Lina

2012-01-01

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.

Biomaterials for periodontal regeneration

PubMed Central

Shue, Li; Yufeng, Zhang; Mony, Ullas

2012-01-01

Periodontal disease is characterized by the destruction of periodontal tissues. Various methods of regenerative periodontal therapy, including the use of barrier membranes, bone replacement grafts, growth factors and the combination of these procedures have been investigated. The development of biomaterials for tissue engineering has considerably improved the available treatment options above. They fall into two broad classes: ceramics and polymers. The available ceramic-based materials include calcium phosphate (eg, tricalcium phosphate and hydroxyapatite), calcium sulfate and bioactive glass. The bioactive glass bonds to the bone with the formation of a layer of carbonated hydroxyapatite in situ. The natural polymers include modified polysaccharides (eg, chitosan,) and polypeptides (collagen and gelatin). Synthetic polymers [eg, poly(glycolic acid), poly(L-lactic acid)] provide a platform for exhibiting the biomechanical properties of scaffolds in tissue engineering. The materials usually work as osteogenic, osteoconductive and osteoinductive scaffolds. Polymers are more widely used as a barrier material in guided tissue regeneration (GTR). They are shown to exclude epithelial downgrowth and allow periodontal ligament and alveolar bone cells to repopulate the defect. An attempt to overcome the problems related to a collapse of the barrier membrane in GTR or epithelial downgrowth is the use of a combination of barrier membranes and grafting materials. This article reviews various biomaterials including scaffolds and membranes used for periodontal treatment and their impacts on the experimental or clinical management of periodontal defect. PMID:23507891

The Role of Synaptopodin in Membrane Protein Diffusion in the Dendritic Spine Neck.

PubMed

Wang, Lili; Dumoulin, Andréa; Renner, Marianne; Triller, Antoine; Specht, Christian G

2016-01-01

The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-specificity of excitatory synapses and to set the scale on which functional changes can occur. Synaptopodin is located predominantly in the neck of dendritic spines, and is thus ideally placed to regulate the exchange of synaptic membrane proteins. The central aim of our study was to assess whether the presence of synaptopodin influences the mobility of membrane proteins in the spine neck and to characterise whether this was due to direct molecular interactions or to spatial constraints that are related to the structural organisation of the neck. Using single particle tracking we have identified a specific effect of synaptopodin on the diffusion of metabotropic mGluR5 receptors in the spine neck. However, super-resolution STORM/PALM imaging showed that this was not due to direct interactions between the two proteins, but that the presence of synaptopodin is associated with an altered local organisation of the F-actin cytoskeleton, that in turn could restrict the diffusion of membrane proteins with large intracellular domains through the spine neck. This study contributes new data on the way in which the spine neck compartmentalises excitatory synapses. Our data complement models that consider the impact of the spine neck as a function of its shape, by showing that the internal organisation of the neck imposes additional physical barriers to membrane protein diffusion.

The Role of Synaptopodin in Membrane Protein Diffusion in the Dendritic Spine Neck

PubMed Central

Wang, Lili; Dumoulin, Andréa; Renner, Marianne; Triller, Antoine; Specht, Christian G.

2016-01-01

The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-specificity of excitatory synapses and to set the scale on which functional changes can occur. Synaptopodin is located predominantly in the neck of dendritic spines, and is thus ideally placed to regulate the exchange of synaptic membrane proteins. The central aim of our study was to assess whether the presence of synaptopodin influences the mobility of membrane proteins in the spine neck and to characterise whether this was due to direct molecular interactions or to spatial constraints that are related to the structural organisation of the neck. Using single particle tracking we have identified a specific effect of synaptopodin on the diffusion of metabotropic mGluR5 receptors in the spine neck. However, super-resolution STORM/PALM imaging showed that this was not due to direct interactions between the two proteins, but that the presence of synaptopodin is associated with an altered local organisation of the F-actin cytoskeleton, that in turn could restrict the diffusion of membrane proteins with large intracellular domains through the spine neck. This study contributes new data on the way in which the spine neck compartmentalises excitatory synapses. Our data complement models that consider the impact of the spine neck as a function of its shape, by showing that the internal organisation of the neck imposes additional physical barriers to membrane protein diffusion. PMID:26840625

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

PubMed Central

Jensen, Victor L; Li, Chunmei; Bowie, Rachel V; Clarke, Lara; Mohan, Swetha; Blacque, Oliver E; Leroux, Michel R

2015-01-01

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. PMID:26392567

Rationalization of reduced penetration of drugs through ceramide gel phase membrane.

PubMed

Paloncýová, Markéta; DeVane, Russell H; Murch, Bruce P; Berka, Karel; Otyepka, Michal

2014-11-25

Since computing resources have advanced enough to allow routine molecular simulation studies of drug molecules interacting with biologically relevant membranes, a considerable amount of work has been carried out with fluid phospholipid systems. However, there is very little work in the literature on drug interactions with gel phase lipids. This poses a significant limitation for understanding permeation through the stratum corneum where the primary pathway is expected to be through a highly ordered lipid matrix. To address this point, we analyzed the interactions of p-aminobenzoic acid (PABA) and its ethyl (benzocaine) and butyl (butamben) esters with two membrane bilayers, which differ in their fluidity at ambient conditions. We considered a dioleoylphosphatidylcholine (DOPC) bilayer in a fluid state and a ceramide 2 (CER2, ceramide NS) bilayer in a gel phase. We carried out unbiased (100 ns long) and biased z-constraint molecular dynamics simulations and calculated the free energy profiles of all molecules along the bilayer normal. The free energy profiles converged significantly slower for the gel phase. While the compounds have comparable affinities for both membranes, they exhibit penetration barriers almost 3 times higher in the gel phase CER2 bilayer. This elevated barrier and slower diffusion in the CER2 bilayer, which are caused by the high ordering of CER2 lipid chains, explain the low permeability of the gel phase membranes. We also compared the free energy profiles from MD simulations with those obtained from COSMOmic. This method provided the same trends in behavior for the guest molecules in both bilayers; however, the penetration barriers calculated by COSMOmic did not differ between membranes. In conclusion, we show how membrane fluid properties affect the interaction of drug-like molecules with membranes.

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

PubMed Central

Groves, J T; Wülfing, C; Boxer, S G

1996-01-01

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8913608

Plasma membrane translocation of a protein needle based on a triple-stranded β-helix motif.

PubMed

Sanghamitra, Nusrat J M; Inaba, Hiroshi; Arisaka, Fumio; Ohtan Wang, Dan; Kanamaru, Shuji; Kitagawa, Susumu; Ueno, Takafumi

2014-10-01

Plasma membrane translocation is challenging due to the barrier of the cell membrane. Contrary to the synthetic cell-penetrating materials, tailed bacteriophages use cell-puncturing protein needles to puncture the cell membranes as an initial step of the DNA injection process. Cell-puncturing protein needles are thought to remain functional in the native phages. In this paper, we found that a bacteriophage T4 derived protein needle of 16 nm length spontaneously translocates through the living cell membrane. The β-helical protein needle (β-PN) internalizes into human red blood cells that lack endocytic machinery. By comparing the cellular uptake of β-PNs with modified surface charge, it is shown that the uptake efficiency is maximum when it has a negative charge corresponding to a zeta potential value of -16 mV. In HeLa cells, uptake of β-PN incorporates endocytosis independent mechanisms with partial macropinocytosis dependence. The endocytosis dependence of the uptake increases when the surface charges of β-PNs are modified to positive or negative. Thus, these results suggest that natural DNA injecting machinery can serve as an inspiration to design new class of cell-penetrating materials with a tailored mechanism.

Resurrecting Inactive Antimicrobial Peptides from the Lipopolysaccharide Trap

PubMed Central

Mohanram, Harini

2014-01-01

Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, “trapped” by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action. PMID:24419338

Xyloglucan, a Plant Polymer with Barrier Protective Properties over the Mucous Membranes: An Overview.

PubMed

Piqué, Núria; Gómez-Guillén, María Del Carmen; Montero, María Pilar

2018-02-27

Disruption of the epithelial barrier function has been recently associated with a variety of diseases, mainly at intestinal level, but also affecting the respiratory epithelium and other mucosal barriers. Non-pharmacological approaches such as xyloglucan, with demonstrated protective barrier properties, are proposed as new alternatives for the management of a wide range of diseases, for which mucosal disruption and, particularly, tight junction alterations, is a common characteristic. Xyloglucan, a natural polysaccharide derived from tamarind seeds, possesses a "mucin-like" molecular structure that confers mucoadhesive properties, allowing xyloglucan formulations to act as a barrier capable of reducing bacterial adherence and invasion and to preserve tight junctions and paracellular flux, as observed in different in vitro and in vivo studies. In clinical trials, xyloglucan has been seen to reduce symptoms of gastroenteritis in adults and children, nasal disorders and dry eye syndrome. Similar mucosal protectors containing reticulated proteins have also been useful for the treatment of irritable bowel syndrome and urinary tract infections. The role of xyloglucan in other disorders with mucosal disruption, such as dermatological or other infectious diseases, deserves further research. In conclusion, xyloglucan, endowed with film-forming protective barrier properties, is a safe non-pharmacological alternative for the management of different diseases, such as gastrointestinal and nasal disorders.

Xyloglucan, a Plant Polymer with Barrier Protective Properties over the Mucous Membranes: An Overview

PubMed Central

Gómez-Guillén, María del Carmen; Montero, María Pilar

2018-01-01

Disruption of the epithelial barrier function has been recently associated with a variety of diseases, mainly at intestinal level, but also affecting the respiratory epithelium and other mucosal barriers. Non-pharmacological approaches such as xyloglucan, with demonstrated protective barrier properties, are proposed as new alternatives for the management of a wide range of diseases, for which mucosal disruption and, particularly, tight junction alterations, is a common characteristic. Xyloglucan, a natural polysaccharide derived from tamarind seeds, possesses a “mucin-like” molecular structure that confers mucoadhesive properties, allowing xyloglucan formulations to act as a barrier capable of reducing bacterial adherence and invasion and to preserve tight junctions and paracellular flux, as observed in different in vitro and in vivo studies. In clinical trials, xyloglucan has been seen to reduce symptoms of gastroenteritis in adults and children, nasal disorders and dry eye syndrome. Similar mucosal protectors containing reticulated proteins have also been useful for the treatment of irritable bowel syndrome and urinary tract infections. The role of xyloglucan in other disorders with mucosal disruption, such as dermatological or other infectious diseases, deserves further research. In conclusion, xyloglucan, endowed with film-forming protective barrier properties, is a safe non-pharmacological alternative for the management of different diseases, such as gastrointestinal and nasal disorders. PMID:29495535

Alport syndrome and Pierson syndrome: Diseases of the glomerular basement membrane.

PubMed

Funk, Steven D; Lin, Meei-Hua; Miner, Jeffrey H

2018-04-16

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5β2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

PubMed Central

Piek, Susannah; Kahler, Charlene M.

2012-01-01

The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism. PMID:23267440

Atomistic Simulations of the pH Induced Functional Rearrangement of Influenza Hemagglutinin

NASA Astrophysics Data System (ADS)

Lin, Xingcheng; Noel, Jeffrey; Wang, Qinghua; Ma, Jianpeng; Onuchic, Jose

Influenza hemagglutinin (HA), a surface glycoprotein responsible for the entry and replication of flu viruses in their host cells, functions by starting a dramatic conformational rearrangement, which leads to a fusion of the viral and endosomal membranes. It has been claimed that a loop-to-coiled-coil transition of the B-loop domain of HA drives the HA-induced membrane fusion. On the lack of dynamical details, however, the microscopic picture for this proposed ``spring-loaded'' movement is missing. To elaborate on the transition of the B-loop, we performed a set of unbiased all-atom molecular dynamics simulations of the full B-loop structure with the CHARMM36 force field. The complete free-energy profile constructed from our simulations reveals a slow transition rate for the B-loop that is incompatible with a downhill process. Additionally, our simulations indicate two potential sources of kinetic traps in the structural switch of the B-loop: Desolvation barriers and non-native secondary structure formation. The slow timescale of the B-loop transition also confirms our previous discovery from simulations using a coarse-grained structure-based model, which identified two competitive pathways both with a slow B-loop transition for HA to guide the membrane fusion.

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

PubMed Central

Frezza, Ludivine; Sandtner, Walter

2013-01-01

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing. PMID:24127524

Dense, layered membranes for hydrogen separation

DOEpatents

Roark, Shane E.; MacKay, Richard; Mundschau, Michael V.

2006-02-21

This invention provides hydrogen-permeable membranes for separation of hydrogen from hydrogen-containing gases. The membranes are multi-layer having a central hydrogen-permeable layer with one or more catalyst layers, barrier layers, and/or protective layers. The invention also relates to membrane reactors employing the hydrogen-permeable membranes of the invention and to methods for separation of hydrogen from a hydrogen-containing gas using the membranes and reactors. The reactors of this invention can be combined with additional reactor systems for direct use of the separated hydrogen.

Mechanics, morphology, and mobility in stratum corneum membranes

NASA Astrophysics Data System (ADS)

Olmsted, Peter; Das, Chinmay; Noro, Massimo

2012-02-01

The stratum corneum is the outermost layer of skin, and serves as a protective barrier against external agents, and to control moisture. It comprises keratin bodies (corneocytes) embedded in a matrix of lipid bilayers. Unlike the more widely studied phospholipid bilayers, the SC bilayers are typically in a gel-like state. Moreover, the SC membrane composition is radically different from more fluid counterparts: it comprises single tailed fatty acids, ceramides, and cholesterol; with many distinct ceramides possessing different lengths of tails, and always with two tails of different lengths. I will present insight from computer simulations into the morphology, mechanical properties, and diffusion (barrier) properties of these highly heterogeneous membranes. Our results provide some clue as to the design principles for the SC membrane, and is an excellent example of the use of wide polydispersity by natural systems.

Ion exchange membrane textile bioreactor as a new alternative for drinking water denitrification.

PubMed

Berdous, Dalila; Akretche, Djamal-Eddine; Abderahmani, Ahmed; Berdous, Sakina; Meknaci, Rima

2014-06-01

This work enters in the optics of the denitrification of a polluted water by two membrane techniques, the Donnan dialysis (DD) and the ion exchange membrane bioreactor (IEMB), using a conventional barrier, composed by an anion exchange membrane (AEM), and a hybrid barrier, where the AEM is combined to an anion exchange textile (AET). The effects of the hydrodynamic factor and the nature of the carbon source on the transfer and the reduction of nitrate ions were studied. The study results obtained through the DD showed the effectiveness of the hybrid barrier in the recovery and concentration of nitrate ions. This was also recorded during denitrification by the hybrid process, called the ion exchange membrane textile bioreactor (IEMTB), with a significant reduction of nitrates, compared to IEMB, due to the efficiency of the Pseudomonas aeruginosa biofilm formed at the surface of the AET. Here, the permselectivity of the membrane and the good bioreduction of the pollutants are no longer major conditions to the better performance of the process. The application of IEMTB in the denitrification of groundwater, having a nitrate concentration of 96.67 ppm, shows a total reduction of nitrate ions without changing the quality of the water. Indeed, the analysis of the recovered water, or yet the treated water, shows the absence of the bacterium by-products and concentrations in the nitrates and nitrites which are, respectively, equal to 0.02±0.01 ppm, and inferiors to the detection limit (<0.02 ppm).

Polymeric membranes for guided bone regeneration.

PubMed

Gentile, Piergiorgio; Chiono, Valeria; Tonda-Turo, Chiara; Ferreira, Ana M; Ciardelli, Gianluca

2011-10-01

In this review, different barrier membranes for guided bone regeneration (GBR) are described as a useful surgical technique to enhance bone regeneration in damaged alveolar sites before performing implants and fitting other dental appliances. The GBR procedure encourages bone regeneration through cellular exclusion and avoids the invasion of epithelial and connective tissues that grow at the defective site instead of bone tissue. The barrier membrane should satisfy various properties, such as biocompatibility, non-immunogenicity, non-toxicity, and a degradation rate that is long enough to permit mechanical support during bone formation. Other characteristics such as tissue integration, nutrient transfer, space maintenance and manageability are also of interest. In this review, various non-resorbable and resorbable commercially available membranes are described, based on expanded polytetrafluoroethylene, poly(lactic acid), poly(glycolic acid) and their copolymers. The polyester-based membranes are biodegradable, permit a single-stage procedure, and have higher manageability than non-resorbable membranes; however, they have shown poor biocompatibility. In contrast, membranes based on natural materials, such as collagen, are biocompatible but are characterized by poor mechanical properties and stability due to their early degradation. Moreover, new approaches are described, such as the use of multi-layered, graft-copolymer-based and composite membranes containing osteoconductive ceramic fillers as alternatives to conventional membranes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins

PubMed Central

Li, Yingxue; Lefever, Mark R; Muthu, Dhanasekaran; Bidlack, Jean M; Bilsky, Edward J; Polt, Robin

2012-01-01

Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood–brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates. PMID:22300099

Transmembrane proteins of tight junctions.

PubMed

Chiba, Hideki; Osanai, Makoto; Murata, Masaki; Kojima, Takashi; Sawada, Norimasa

2008-03-01

Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.

Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport

PubMed Central

Sherman, David J.; Lazarus, Michael B.; Murphy, Lea; Liu, Charles; Walker, Suzanne; Ruiz, Natividad; Kahne, Daniel

2014-01-01

The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site. PMID:24639492

Practical Application of Sheet Lead for Sound Barriers.

ERIC Educational Resources Information Center

Lead Industries Association, New York, NY.

Techniques for improving sound barriers through the use of lead sheeting are described. To achieve an ideal sound barrier a material should consist of the following properties--(1) high density, (2) freedom from stiffness, (3) good damping capacity, and (4) integrity as a non-permeable membrane. Lead combines these desired properties to a greater…

A Mechanistic Study of Arsenic (III) Rejection by Reverse Osmosis and Nanofiltration Membranes

ERIC Educational Resources Information Center

Suzuki, Tasuma

2009-01-01

Reverse osmosis/nanofiltration (RO/NF) membranes are capable to provide an effective barrier for a wide range of contaminants (including disinfection by-products precursors) in a single treatment step. However, solute rejection mechanisms by RO/NF membranes are not well understood. The lack of mechanistic information arises from experimental…

Electromechanically Actuated Multifunctional Wireless Auxetic Device for Wound Management.

PubMed

Mir, Mariam; Ansari, Umar; Ali, Murtaza Najabat; Iftikhar, Muhammad Hassan Ul; Qayyum, Faisal

2017-01-01

The design and fabrication of a wound healing device for chronic wounds, with multiple functions for controlled drug delivery and exudate removal, has been described in this paper. The structural features have been machined and modified through laser cutting in a biocompatible polymer cast. Miniaturized versions of electronically actuated (lead-screw and pulley) mechanisms are used for the specific purpose of controlled drug delivery. These mechanisms have been studied and tested, being controlled through a microcontroller setup. An auxetic polymeric barrier membrane has been used for restricting the drug quantities administered. Drug delivery mechanisms are powered wirelessly, through an external, active RF component; this communicates with a passive component that is buried inside the wound healing device. The exudate removal efficiency of the device has been assessed through several simple tests using simulated wound exudate. It has been found that reasonably precise quantities of drug dosages to be administered to the wound site can be controlled through both drug delivery mechanisms; however, the lead-screw mechanism provides a better control of auxetic barrier membrane actuation and hence controlled drug delivery. We propose that this device can have potential clinical significance in controlled drug delivery and exudate removal in the management of chronic wounds.

Evaluation of possible reasons for the low phenylalanine ammonia lyase activity in cellulose nitrate membrane microcapsules.

PubMed

Habibi-Moini, S; D'mello, A P

2001-03-14

Microencapsulated phenylalanine ammonia lyase (PAL) exhibits a marked reduction in activity compared to the activity of the free enzyme in pH 8.5 Tris buffer. The purpose of this investigation was to evaluate the contribution of incomplete entrapment, the internal environment of cellulose nitrate membrane microcapsules, the diffusional barrier of the membrane and the microcapsulation process to the low activity of encapsulated PAL. A solution of PAL and 10% w/v hemoglobin was incorporated into cellulose nitrate membrane microcapsules. Hemoglobin incorporation was used as a surrogate marker of PAL entrapment. Using 14C hemoglobin, the encapsulation efficiency was determined to be 70% and suggested that incomplete entrapment might partially account for the low activity of encapsulated PAL. The effect of the internal environment of the microcapsule (10% hemoglobin solution) on PAL activity was evaluated by comparing enzyme activity in 10% w/v hemoglobin solution and pH 8.5 Tris buffer. Similar K(M) and V(max) values of PAL in the two media indicated that the internal environment of the microcapsule did not contribute to the reduction in activity of the encapsulated enzyme. The contribution of a membrane diffusional barrier was determined by breaking the putative barrier and measuring PAL activity in intact and broken microcapsules. Similar activity of PAL in these two conditions is evidence for the lack of a diffusional barrier. The effect of the microencapsulation process on PAL activity was evaluated by comparing K(M) and V(max) of free and encapsulated PAL. Similar K(M) values in these two media suggested that the process did not affect the conformation of PAL. However, encapsulated PAL had a 50% lower V(max) value compared to free PAL, which showed that the microencapsulation process deactivated a substantial proportion of the enzyme.

Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins

PubMed Central

Yang, Nicole J.; Hinner, Marlon J.

2016-01-01

The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome. PMID:25560066

An evaluation of bacterial contamination of barriers used in periapical tissue regeneration: Part 1--Bacterial adherence.

PubMed

Sharma, Priya; Mickel, André K; Chogle, Sami; Sharma, Prem Nath; Han, Yiping W; Jones, Jefferson J

2008-02-01

To compare the adherence of Prevotella melaninogenica and Enterococcus faecalis to 3 guided tissue regeneration membranes: Atrisorb, Lambone, and OsseoQuest. It was hypothesized that OsseoQuest would show increased bacterial adherence compared to Lambone and Atrisorb. The barriers were suspended in trypticase soy broth containing an inoculum of either P melaninogenica or E faecalis. The samples were incubated under appropriate conditions for 6, 24, and 48 hours. Following incubation, each membrane was mixed in fresh media in a vortex machine to dislodge adherent bacteria. The vortexed media was quantitatively assessed using serial dilutions for viable cell count. E faecalis exhibited higher adherence compared to P melaninogenica with time. Of the membranes tested, Lambone displayed the least bacterial adherence. An analysis of the results indicated that bacterial adherence was time-dependent for all membranes. Membrane structure, chemical configuration, hydrophobicity, and bacterial cell surface structure were suggested as factors contributing to variance in bacterial adherence.

Ultrathin epitaxial barrier layer to avoid thermally induced phase transformation in oxide heterostructures

DOE PAGES

Baek, David J.; Lu, Di; Hikita, Yasuyuki; ...

2016-12-22

Incorporating oxides with radically different physical and chemical properties into heterostructures offers tantalizing possibilities to derive new functions and structures. Recently, we have fabricated freestanding 2D oxide membranes using the water-soluble perovskite Sr 3Al 2O 6 as a sacrificial buffer layer. Here, with atomic-resolution spectroscopic imaging, we observe that direct growth of oxide thin films on Sr 3Al 2O 6 can cause complete phase transformation of the buffer layer, rendering it water-insoluble. More importantly, we demonstrate that an ultrathin SrTiO 3 layer can be employed as an effective barrier to preserve Sr 3Al 2O 6 during subsequent growth, thus allowingmore » its integration in a wider range of oxide heterostructures.« less

Determination of Urea Permeability in Red Cells by Minimum Method

PubMed Central

Sha'afi, R. I.; Rich, G. T.; Mikulecky, D. C.; Solomon, A. K.

1970-01-01

A new method has been developed for measuring the permeability coefficient, ω, of small nonelectrolytes. The method depends upon a mathematical analysis of the time course of cell volume changes in the neighborhood of the minimum volume following addition of a permeating solute to an isosmolal buffer. Coefficients determined by the minimum volume method agree with those obtained using radioactive tracers. ω for urea in human red cells was found to decrease as the volume flow, Jv, into the cell increased. Such behavior is entirely unexpected for a single uniform rate-limiting barrier on the basis of the linear phenomenological equations derived from irreversible thermodynamics. However, the present findings are consonant with a complex membrane system consisting of a tight barrier on the outer face of the human red cell membrane and a somewhat less restrictive barrier behind it closer to the inner membrane face. A theoretical analysis of such a series model has been made which makes predictions consistent with the experimental findings. PMID:5435779

Hormones and the blood-brain barrier.

PubMed

Hampl, Richard; Bičíková, Marie; Sosvorová, Lucie

2015-03-01

Hormones exert many actions in the brain, and brain cells are also hormonally active. To reach their targets in brain structures, hormones must overcome the blood-brain barrier (BBB). The BBB is a unique device selecting desired/undesired molecules to reach or leave the brain, and it is composed of endothelial cells forming the brain vasculature. These cells differ from other endothelial cells in their almost impermeable tight junctions and in possessing several membrane structures such as receptors, transporters, and metabolically active molecules, ensuring their selection function. The main ways how compounds pass through the BBB are briefly outlined in this review. The main part concerns the transport of major classes of hormones: steroids, including neurosteroids, thyroid hormones, insulin, and other peptide hormones regulating energy homeostasis, growth hormone, and also various cytokines. Peptide transporters mediating the saturable transport of individual classes of hormones are reviewed. The last paragraph provides examples of how hormones affect the permeability and function of the BBB either at the level of tight junctions or by various transporters.

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

PubMed

Skjørringe, Tina; Burkhart, Annette; Johnsen, Kasper Bendix; Moos, Torben

2015-01-01

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology

PubMed Central

Skjørringe, Tina; Burkhart, Annette; Johnsen, Kasper Bendix; Moos, Torben

2015-01-01

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail. PMID:26106291

Celiac Disease: Role of the Epithelial Barrier.

PubMed

Schumann, Michael; Siegmund, Britta; Schulzke, Jörg D; Fromm, Michael

2017-03-01

In celiac disease (CD) a T-cell-mediated response to gluten is mounted in genetically predisposed individuals, resulting in a malabsorptive enteropathy histologically highlighted by villous atrophy and crypt hyperplasia. Recent data point to the epithelial layer as an under-rated hot spot in celiac pathophysiology to date. This overview summarizes current functional and genetic evidence on the role of the epithelial barrier in CD, consisting of the cell membranes and the apical junctional complex comprising sealing as well as ion and water channel-forming tight junction proteins and the adherens junction. Moreover, the underlying mechanisms are discussed, including apoptosis of intestinal epithelial cells, biology of intestinal stem cells, alterations in the apical junctional complex, transcytotic uptake of gluten peptides, and possible implications of a defective epithelial polarity. Current research is directed toward new treatment options for CD that are alternatives or complementary therapeutics to a gluten-free diet. Thus, strategies to target an altered epithelial barrier therapeutically also are discussed.

Actin dynamics regulate immediate PAR-2-dependent responses to acute epidermal permeability barrier abrogation.

PubMed

Roelandt, Truus; Heughebaert, Carol; Verween, Gunther; Giddelo, Christina; Verbeken, Gilbert; Pirnay, Jean-Paul; Devos, Daniel; Crumrine, Debra; Roseeuw, Diane; Elias, Peter M; Hachem, Jean-Pierre

2011-02-01

Lamellar body (LB) secretion and terminal differentiation of stratum granulosum (SG) cells are signaled by both protease activated receptor-2 (PAR-2) and caveolin-1 (cav-1). To address the early dynamics of LB secretion, we examined cytoskeletal remodeling of keratinocytes in 3 mouse models following acute barrier abrogation: hairless mice, PAR-2 knockout (-/-) and cav-1 -/-. Under basal conditions, globular (G)-actin accumulates in SG cells cytosol, while filamentous (F)-actin is restricted to peri-membrane domains. Barrier abrogation induces the apical movement of F-actin and the retreat of the SG-G-actin front, paralleled by upstream cytoskeletal kinases activation. This phenomenon was both enhanced by PAR-2 agonist, and inhibited by cytochalasin-D and in PAR-2 knockout mice. We found that plasma membrane conformational changes causing LB secretion are controlled by PAR-2-dependent cytoskeletal rearrangements. We next addressed the interaction dynamics between cytoskeleton and plasma membrane following PAR-2-induced actin stress fiber formation in both cav-1 -/- and wildtype cells. Actin stress fiber formation is increased in cav-1 -/- cells prior to and following PAR-2 agonist peptide-treatment, while absence of cav-1 inhibits E-cadherin-mediated cell-to-cell adhesion. PAR-2 drives cytoskeletal/plasma membrane dynamics that regulate early LB secretion following barrier abrogation, stress fiber formation and keratinocyte adhesion. Copyright © 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The Role of Nanoparticle Surface Functionality in the Disruption of Model Cell Membranes

PubMed Central

Moghadam, Babak Y.; Hou, Wen-Che; Corredor, Charlie; Westerhoff, Paul; Posner, Jonathan D.

2012-01-01

Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of engineered nanomaterials (ENMs) with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assess the effect of varying the surface functionality and concentration of 10 nm-diameter gold (Au) and titanium dioxide (TiO2) ENMs on the disruption of negatively charged lipid bilayer vesicles (liposomes) using a dye leakage assay. Our findings show that Au ENMs having both positive and negative surface charge induce leakage that reaches a steady state after several hours. Positively charged particles with identical surface functionality and different core composition show similar leakage effects and result in faster and greater leakage than negatively charged particles, which suggests that surface functionality, not particle core composition, is a critical factor in determining the interaction between ENMs and lipid bilayers. The results suggest that particles permanently adsorb to bilayers and that only one positively charged particle is required to disrupt a liposome and trigger leakage of its entire contents in contrast to mellitin molecules, the most widely studied membrane lytic peptide, which requires hundred of molecules to generate leakage. PMID:22921268

Graphene Membranes for Atmospheric Pressure Photoelectron Spectroscopy.

PubMed

Weatherup, Robert S; Eren, Baran; Hao, Yibo; Bluhm, Hendrik; Salmeron, Miquel B

2016-05-05

Atmospheric pressure X-ray photoelectron spectroscopy (XPS) is demonstrated using single-layer graphene membranes as photoelectron-transparent barriers that sustain pressure differences in excess of 6 orders of magnitude. The graphene serves as a support for catalyst nanoparticles under atmospheric pressure reaction conditions (up to 1.5 bar), where XPS allows the oxidation state of Cu nanoparticles and gas phase species to be simultaneously probed. We thereby observe that the Cu(2+) oxidation state is stable in O2 (1 bar) but is spontaneously reduced under vacuum. We further demonstrate the detection of various gas-phase species (Ar, CO, CO2, N2, O2) in the pressure range 10-1500 mbar including species with low photoionization cross sections (He, H2). Pressure-dependent changes in the apparent binding energies of gas-phase species are observed, attributable to changes in work function of the metal-coated grids supporting the graphene. We expect atmospheric pressure XPS based on this graphene membrane approach to be a valuable tool for studying nanoparticle catalysis.

The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b

PubMed Central

Cao, Chunzhang; Gao, Yamei; Li, Yang; Antalis, Toni M.; Castellino, Francis J.; Zhang, Li

2010-01-01

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti­inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor–1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the γ-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase–1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities. PMID:20458145

DMSO Induces Dehydration near Lipid Membrane Surfaces

PubMed Central

Cheng, Chi-Yuan; Song, Jinsuk; Pas, Jolien; Meijer, Lenny H.H.; Han, Songi

2015-01-01

Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (XDMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at XDMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At XDMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw. PMID:26200868

Inactivation of Escherichia coli and Staphylococcus aureus on contaminated perilla leaves by Dielectric Barrier Discharge (DBD) plasma treatment.

PubMed

Ji, Sang Hye; Ki, Se Hoon; Ahn, Ji Ho; Shin, Jae Ho; Hong, Eun Jeong; Kim, Yun Ji; Choi, Eun Ha

2018-04-02

This study focused on sterilization methods for the reduction of microorganisms on perilla leaves by cylinder type Dielectric Barrier Discharge (DBD) plasma with underwater bubbler treatment. S. aureus and E. coli in a suspension were reduced to less than 3.4 and 0.5 log CFU/ml after the plasma treatment for 3 min, respectively. On the perilla leaves, they were also reduced to 4.8 and 1.6 log CFU/ml after the plasma treatment, respectively. The S. aureus and E. coli bacterial cell wall was damaged by the plasma treatment evident by scanning electron microscopic analysis. The observed infrared bands of the FTIR spectra demonstrated changes in protein, lipid, polysaccharide, polyphosphate group and other carbohydrate functionalities of plasma treated bacteria and untreated bacterial cell membranes. The degradation of the constituent bonds of the bacterial cell membrane by RONS generated from plasma destroys the DNA, RNA, and proteins within the cell, and may eventually cause cell death. In this study, H 2 O 2 (13.68 μM) and NO 3 (138 μM), which are the main factors generated by plasma, proved to have a bactericidal effect by inducing lipid peroxidation of bacterial cell membranes. In conclusion, cylinder type DBD plasma with underwater bubbler can be used as an environmentally friendly food disinfection device in cleaning processes of the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

Basement Membrane Defects in Genetic Kidney Diseases

PubMed Central

Chew, Christine; Lennon, Rachel

2018-01-01

The glomerular basement membrane (GBM) is a specialized structure with a significant role in maintaining the glomerular filtration barrier. This GBM is formed from the fusion of two basement membranes during development and its function in the filtration barrier is achieved by key extracellular matrix components including type IV collagen, laminins, nidogens, and heparan sulfate proteoglycans. The characteristics of specific matrix isoforms such as laminin-521 (α5β2γ1) and the α3α4α5 chain of type IV collagen are essential for the formation of a mature GBM and the restricted tissue distribution of these isoforms makes the GBM a unique structure. Detailed investigation of the GBM has been driven by the identification of inherited abnormalities in matrix proteins and the need to understand pathogenic mechanisms causing severe glomerular disease. A well-described hereditary GBM disease is Alport syndrome, associated with a progressive glomerular disease, hearing loss, and lens defects due to mutations in the genes COL4A3, COL4A4, or COL4A5. Other proteins associated with inherited diseases of the GBM include laminin β2 in Pierson syndrome and LMX1B in nail patella syndrome. The knowledge of these genetic mutations associated with GBM defects has enhanced our understanding of cell–matrix signaling pathways affected in glomerular disease. This review will address current knowledge of GBM-associated abnormalities and related signaling pathways, as well as discussing the advances toward disease-targeted therapies for patients with glomerular disease. PMID:29435440

Epithelial Microvilli Establish an Electrostatic Barrier to Microbial Adhesion

PubMed Central

Bennett, Kaila M.; Walker, Sharon L.

2014-01-01

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli (“microvillus-minus,” or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions. PMID:24778113

Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application.

PubMed

Carter, Princeton; Rahman, Shekh M; Bhattarai, Narayan

2016-01-01

Guided tissue regeneration (GTR) is a widely used method in dental surgical procedures that utilizes a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells at the sites having insufficient gingiva. Commercial GTR membranes are typically composed of synthetic polymers that have had mild clinical success mostly because of their lack of proper bioactivity and appropriate degradation profile. In this study, a natural polymer, aloe vera was blended with polycaprolactone (PCL) to create nanofibrous GTR membranes by electrospinning. Aloe vera has proven anti-inflammatory properties and enhances the regeneration of periodontium tissues. PCL, a synthetic polymer, is well known to produce miscible polyblends nanofibers with natural polymers. Nanofibrous membranes with varying composition of PCL to aloe vera were fabricated, and several physicochemical and biological properties, such as fiber morphology, wettability, chemical structure, mechanical strength, and cellular compatibility of the membranes were analyzed. PCL/aloe vera membranes with ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in aqueous solutions. Experimental results, using cell viability assay and cell attachment observation, showed that the nanofibrous membranes support 3T3 cell viability and could be a potential candidate for GTR therapy.

Abnormal N-Glycosylation of a Novel Missense Creatine Transporter Mutant, G561R, Associated with Cerebral Creatine Deficiency Syndromes Alters Transporter Activity and Localization.

PubMed

Uemura, Tatsuki; Ito, Shingo; Ohta, Yusuke; Tachikawa, Masanori; Wada, Takahito; Terasaki, Tetsuya; Ohtsuki, Sumio

2017-01-01

Cerebral creatine deficiency syndromes (CCDSs) are caused by loss-of-function mutations in creatine transporter (CRT, SLC6A8), which transports creatine at the blood-brain barrier and into neurons of the central nervous system (CNS). This results in low cerebral creatine levels, and patients exhibit mental retardation, poor language skills and epilepsy. We identified a novel human CRT gene missense mutation (c.1681 G>C, G561R) in Japanese CCDSs patients. The purpose of the present study was to evaluate the reduction of creatine transport in G561R-mutant CRT-expressing 293 cells, and to clarify the mechanism of its functional attenuation. G561R-mutant CRT exhibited greatly reduced creatine transport activity compared to wild-type CRT (WT-CRT) when expressed in 293 cells. Also, the mutant protein is localized mainly in intracellular membrane fraction, while WT-CRT is localized in plasma membrane. Western blot analysis revealed a 68 kDa band of WT-CRT protein in plasma membrane fraction, while G561R-mutant CRT protein predominantly showed bands at 55, 110 and 165 kDa in crude membrane fraction. The bands of both WT-CRT and G561R-mutant CRT were shifted to 50 kDa by N-glycosidase treatment. Our results suggest that the functional impairment of G561R-mutant CRT was probably caused by incomplete N-linked glycosylation due to misfolding during protein maturation, leading to oligomer formation and changes of cellular localization.

Regulated traffic of anion transporters in mammalian Brunner's glands: a role for water and fluid transport.

PubMed

Collaco, Anne M; Jakab, Robert L; Hoekstra, Nadia E; Mitchell, Kisha A; Brooks, Amos; Ameen, Nadia A

2013-08-01

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.

DIRECTIONAL FLUID TRANSPORT ACROSS ORGAN-BLOOD BARRIERS: PHYSIOLOGY AND CELL BIOLOGY

PubMed Central

Caceres, Paulo S.; Benedicto, Ignacio; Lehmann, Guillermo L.; Rodriguez-Boulan, Enrique J.

2018-01-01

Directional fluid flow is an essential process for embryo development as well as for organ and organism homeostasis. Here, we review the diverse structure of various organ-blood barriers, the driving forces, transporters and polarity mechanisms that regulate fluid transport across them, focusing on kidney-, eye- and brain-blood barriers. We end by discussing how cross-talk between barrier epithelial and endothelial cells, perivascular cells and basement membrane signaling contribute to generate and maintain organ-blood barriers. PMID:28003183

EMMPRIN Modulates Epithelial Barrier Function through a MMP–Mediated Occludin Cleavage

PubMed Central

Huet, Eric; Vallée, Benoit; Delbé, Jean; Mourah, Samia; Prulière-Escabasse, Virginie; Tremouilleres, Magali; Kadomatsu, Kenji; Doan, Serge; Baudouin, Christophe; Menashi, Suzanne; Gabison, Eric E.

2011-01-01

Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure. PMID:21777561

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier*

PubMed Central

Argüeso, Pablo; Guzman-Aranguez, Ana; Mantelli, Flavio; Cao, Zhiyi; Ricciuto, Jessica; Panjwani, Noorjahan

2009-01-01

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins. PMID:19556244

In vitro and in vivo assessment of the effect of Laurus novocanariensis oil and essential oil in human skin.

PubMed

Viciolle, E; Castilho, P; Rosado, C

2012-12-01

Laurus novocanariensis is an endemic plant from the Madeira Island forest that derives a fatty oil, with a strong spicy odour, from its berries that has been used for centuries in traditional medicine to treat skin ailments. This work aimed to investigate the effect of the application of both the oil and its essential oil on normal skin, to assess their safety and potential benefits. Diffusion studies with Franz cells using human epidermal membranes were conducted. The steady-state fluxes of two model molecules through untreated skin were compared with those obtained after a 2-h pre-treatment with either the oil or the essential oil. Additionally, eleven volunteers participated in the in vivo study that was conducted on the forearm and involved daily application of the oil for 5 days. Measurements were performed every day in the treated site with bioengineering methods that measure erythema, irritation and loss of barrier function. Slightly higher steady-state fluxes were observed for both the lipophilic and the hydrophilic molecule when the epidermal membranes were pre-treated. Nevertheless, such differences had no statistical significance, which seems to confirm that neither the oil nor the essential oil impaired the epidermal barrier. Results collected with the Chromameter, the Laser Doppler Flowmeter and the visual scoring are in agreement with those established in the in vitro study. They indicate that the repeated application of the oil did not cause erythema, because the results observed in the first day of the study were maintained throughout the week. Application of the oil did not affect the skin barrier function, because the transepidermal water loss remained constant throughout the study. The stratum corneum hydration was slightly reduced on days 4 and 5. This work shows that both the oil and the essential oil were well tolerated by the skin and did not cause significant barrier impairment or irritation. © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Guided bone regeneration produced by new mineralized and reticulated collagen membranes in critical-sized rat calvarial defects

PubMed Central

Leitão, Renata FC; Figueiró, Sônia D; Góes, Júlio C; Lima, Vilma; Silveira, Charles O; Brito, Gerly AC

2015-01-01

The aim of this study was to evaluate the bone regenerative effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen membranes in critical-sized defects on rat calvarias. Bone calvarial defects were induced in Wistar rats, which were then divided into five groups: a sham group; a control group, which received a commercial membrane; and GA, 25GA, and 75GA groups, which received one of three different polyanionic collagen membranes mineralized by 0, 25, or 75 hydroxyapatite cycles and then cross-linked by GA. Bone formation was evaluated based on digital radiography and computerized tomography. Histological analyses were performed 4 and 12 weeks after the surgical procedure to observe bone formation, membrane resorption, and fibrous tissue surrounding the membranes. Measurement of myeloperoxidase activity, tumor necrosis factor alpha, and interleukin 1beta production was performed 24 h after surgery. The percentage of new bone formation in the GA, 25GA, and 75GA groups was higher compared with the control and sham groups. In the GA and 25 GA groups, the membranes were still in place and were contained in a thick fibrous capsule after 12 weeks. No significant difference was found among the groups regarding myeloperoxidase activity and interleukin 1beta levels, although the GA, 25GA, and 75GA groups presented decreased levels of tumor necrosis factor alpha compared with the control group. These new GA cross-linked membranes accelerated bone healing of the calvarium defects and did not induce inflammation. In addition, unlike the control membrane, the experimental membranes were not absorbed during the analyzed period, so they may offer advantages in large bone defects where prolonged membrane barrier functions are desirable. PMID:25245073

Nanosized CaP-silk fibroin-PCL-PEG-PCL/PCL based bilayer membranes for guided bone regeneration.

PubMed

Türkkan, Sibel; Pazarçeviren, A Engin; Keskin, Dilek; Machin, Nesrin E; Duygulu, Özgür; Tezcaner, Ayşen

2017-11-01

Guided bone regeneration (GBR) concept has been developed to prevent the formation of non-functional scar tissue layer on defect site by undertaking barrier role. In this study, a new bilayer membrane which consisted of one layer of electrospun silk fibroin/PCL-PEG-PCL incorporating nanocalcium phosphate (SPCA) 1 and one layer of PCL membrane was developed for GBR. To improve the osteoconductivity of membranes, nanosized calcium phosphate particles synthesized by Flame Spray Pyrolysis method were incorporated into membranes at 10% (wt) (SPCA10) and 20% (wt) (SPCA20) of the polymer content. The structural and chemical analyses revealed the well-integrated two layers of membranes with a total thickness of ca 100μm. In the regenerative layer, the highly porous mesh structure had a thickness of 12.6μm with randomly oriented fibers having diameters around 760nm, and nanoparticles dispersed homogenously. The mechanical test results showed remarkable improvement on the tensile strength of membranes with incorporation of nanoparticles. Higher water affinity of nanoCaP included membranes was proved by lower contact angle values and higher percent water uptake capacity. Biomineralization assay revealed that nucleation and growth of apatites around fibers of SPCA10 and SPCA20 were apparent while on SPCA0 apatite minerals were barely detected after 10days. Human dental pulp stem cells (DPSC) were seeded on electrospun layer of the bilayer membranes for biocompatibility and osteo-compatibility study. Increasing nanoCaP amount resulted in higher cell adhesion, proliferation, ALP activity and calcium deposition on membranes. These overall results confirmed the biocompatibility and potential applicability of proposed membranes for GBR treatments. Copyright © 2017 Elsevier B.V. All rights reserved.

Restricted ADP movement in cardiomyocytes: Cytosolic diffusion obstacles are complemented with a small number of open mitochondrial voltage-dependent anion channels.

PubMed

Simson, Päivo; Jepihhina, Natalja; Laasmaa, Martin; Peterson, Pearu; Birkedal, Rikke; Vendelin, Marko

2016-08-01

Adequate intracellular energy transfer is crucial for proper cardiac function. In energy starved failing hearts, partial restoration of energy transfer can rescue mechanical performance. There are two types of diffusion obstacles that interfere with energy transfer from mitochondria to ATPases: mitochondrial outer membrane (MOM) with voltage-dependent anion channel (VDAC) permeable to small hydrophilic molecules and cytoplasmatic diffusion barriers grouping ATP-producers and -consumers. So far, there is no method developed to clearly distinguish the contributions of cytoplasmatic barriers and MOM to the overall diffusion restriction. Furthermore, the number of open VDACs in vivo remains unknown. The aim of this work was to establish the partitioning of intracellular diffusion obstacles in cardiomyocytes. We studied the response of mitochondrial oxidative phosphorylation of permeabilized rat cardiomyocytes to changes in extracellular ADP by recording 3D image stacks of NADH autofluorescence. Using cell-specific mathematical models, we determined the permeability of MOM and cytoplasmatic barriers. We found that only ~2% of VDACs are accessible to cytosolic ADP and cytoplasmatic diffusion barriers reduce the apparent diffusion coefficient by 6-10×. In cardiomyocytes, diffusion barriers in the cytoplasm and by the MOM restrict ADP/ATP diffusion to similar extents suggesting a major role of both barriers in energy transfer and other intracellular processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hybrid lipid-based nanostructures

NASA Astrophysics Data System (ADS)

Dayani, Yasaman

Biological membranes serve several important roles, such as structural support of cells and organelles, regulation of ionic and molecular transport, barriers to non-mediated transport, contact between cells within tissues, and accommodation of membrane proteins. Membrane proteins and other vital biomolecules incorporated into the membrane need a lipid membrane to function. Due to importance of lipid bilayers and their vital function in governing many processes in the cell, the development of various models as artificial lipid membranes that can mimic cell membranes has become a subject of great interest. Using different models of artificial lipid membranes, such as liposomes, planar lipid bilayers and supported or tethered lipid bilayers, we are able to study many biophysical processes in biological membranes. The ability of different molecules to interact with and change the structure of lipid membranes can be also investigated in artificial lipid membranes. An important application of lipid bilayer-containing interfaces is characterization of novel membrane proteins for high throughput drug screening studies to investigate receptor-drug interactions and develop biosensor systems. Membrane proteins need a lipid bilayer environment to preserve their stability and functionality. Fabrication of materials that can interact with biomolecules like proteins necessitates the use of lipid bilayers as a mimic of cell membranes. The objective of this research is to develop novel hybrid lipid-based nanostructures mimicking biological membranes. Toward this aim, two hybrid biocompatible structures are introduced: lipid bilayer-coated multi-walled carbon nanotubes (MWCNTs) and hydrogel-anchored liposomes with double-stranded DNA anchors. These structures have potential applications in biosensing, drug targeting, drug delivery, and biophysical studies of cell membranes. In the first developed nanostructure, lipid molecules are covalently attached to the surfaces of MWCNTs, and then, using a sonication process, a uniform lipid bilayer that supports the incorporation of membrane proteins is formed. These bilayer-coated carbon nanotubes are highly dispersible and stable in aqueous solution, and they can be used in development of various biosensors and energy producing devices. In the other hybrid nanostructure, the lipid bilayer of a liposome is covalently anchored to a biocompatible poly(ethylene) glycol (PEG) hydrogel core using double-stranded DNA (dsDNA) linkers. Release studies shows that nano-size hydrogel-anchored liposomes are exceptionally stable, and they can be used as biomimetic model membranes that mimic the connectivity between the cytoskeleton and the plasma membrane. After lipid bilayer removal, dsDNA linkers can provide programmable nanogels decorated with oligonucleotides with potential sites for further molecular assembly. These stable nanostructures can be useful for oligonucleotide and drug delivery applications. The developed hydrogel-anchored liposomes are exploited for encapsulation and intracellular delivery of therapeutic peptide. Peptides with anti-cancer properties are successfully encapsulated in hydrogel core of pH-sensitive liposomes during rehydration process. Liposomes release their cargo at acidic pH. Confocal microscopy confirms the intracellular delivery of liposomes through an endocytotic pathway.

Formin like 1 expression is increased on CD4+ T lymphocytes in spontaneous autoimmune uveitis.

PubMed

Degroote, Roxane L; Uhl, Patrizia B; Amann, Barbara; Krackhardt, Angela M; Ueffing, Marius; Hauck, Stefanie M; Deeg, Cornelia A

2017-02-10

The membrane protein expression repertoire of cells changes in course of activation. In equine recurrent uveitis (ERU), a spontaneous autoimmune disease in horses with relapsing and ultimately blinding inner eye inflammation, CD4+ T lymphocytes are the crucial pathogenic cells activated in the periphery directly prior to an inflammatory episode. In order to find relevant changes in the membrane proteome associated to disease, we sorted CD4+ lymphocytes and compared protein abundance from the generated proteome datasets of both healthy horses and ERU cases. We detected formin like 1, a key player in actin dependent cellular processes such as phagocytosis, cell adhesion and cell migration, with significantly higher abundance in the CD4+ cell membrane proteome of horses with ERU. In transmigration experiments, we demonstrated higher migration rate of cells originating from diseased animals connecting formin like 1 to the migratory ability of cells. These findings are the first description of formin like 1 in association to processes involved in migration of inflammatory CD4+ T cells across the blood-retinal barrier in a spontaneous ocular autoimmune disease and suggest formin like 1 to play a role in the molecular mechanisms of ERU disease pathogenesis. Data are available via ProteomeXchange with identifier PXD005384. This comparative proteomic study of membrane proteins of CD4+ T cells identified a novel protein, formin like 1, with expression on the plasma cell membrane of equine CD4+ T cells and a significant change of abundance on CD4+ T cells of horses with a spontaneous autoimmune disease. Functional studies in a cell culture model for transmigration at the blood-retinal barrier (BRB) unraveled a strong impact of formin like 1 on migratory processes of CD4+ T cells across the BRB, a key event in uveitis pathogenesis. These findings provide novel knowledge about changes in the CD4+ immune cell membrane proteome in a spontaneously and naturally occurring autoimmune disease in horses with high relevance for veterinary medicine. Additionally, this model has proven translational quality for human medicine and provides novel proteomic information on autoimmune uveitis in man. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOEpatents

Sansinena, Jose-Maria [Los Alamos, NM; Redondo, Antonio [Los Alamos, NM; Olazabal, Virginia [Los Alamos, NM; Hoffbauer, Mark A [Los Alamos, NM; Akhadov, Elshan A [Los Alamos, NM

2009-12-29

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

2017-09-12

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

2017-07-18

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

The Long and Viscous Road: Uncovering Nuclear Diffusion Barriers in Closed Mitosis

PubMed Central

Zavala, Eder; Marquez-Lago, Tatiana T.

2014-01-01

Diffusion barriers are effective means for constraining protein lateral exchange in cellular membranes. In Saccharomyces cerevisiae, they have been shown to sustain parental identity through asymmetric segregation of ageing factors during closed mitosis. Even though barriers have been extensively studied in the plasma membrane, their identity and organization within the nucleus remains poorly understood. Based on different lines of experimental evidence, we present a model of the composition and structural organization of a nuclear diffusion barrier during anaphase. By means of spatial stochastic simulations, we propose how specialised lipid domains, protein rings, and morphological changes of the nucleus may coordinate to restrict protein exchange between mother and daughter nuclear lobes. We explore distinct, plausible configurations of these diffusion barriers and offer testable predictions regarding their protein exclusion properties and the diffusion regimes they generate. Our model predicts that, while a specialised lipid domain and an immobile protein ring at the bud neck can compartmentalize the nucleus during early anaphase; a specialised lipid domain spanning the elongated bridge between lobes would be entirely sufficient during late anaphase. Our work shows how complex nuclear diffusion barriers in closed mitosis may arise from simple nanoscale biophysical interactions. PMID:25032937

Controlling the hydration of the skin though the application of occluding barrier creams

PubMed Central

Sparr, Emma; Millecamps, Danielle; Isoir, Muriel; Burnier, Véronique; Larsson, Åsa; Cabane, Bernard

2013-01-01

The skin is a barrier membrane that separates environments with profoundly different water contents. The barrier properties are assured by the outer layer of the skin, the stratum corneum (SC), which controls the transepidermal water loss. The SC acts as a responding membrane, since its hydration and permeability vary with the boundary condition, which is the activity of water at the outer surface of the skin. We show how this boundary condition can be changed by the application of a barrier cream that makes a film with a high resistance to the transport of water. We present a quantitative model that predicts hydration and water transport in SC that is covered by such a film. We also develop an experimental method for measuring the specific resistance to water transport of films made of occluding barrier creams. Finally, we combine the theoretical model with the measured properties of the barrier creams to predict how a film of cream changes the activity of water at the outer surface of the SC. Using the known variations of SC permeability and hydration with the water activity in its environment (i.e. the relative humidity), we can thus predict how a film of barrier cream changes SC hydration. PMID:23269846

Controlling the hydration of the skin though the application of occluding barrier creams.

PubMed

Sparr, Emma; Millecamps, Danielle; Isoir, Muriel; Burnier, Véronique; Larsson, Åsa; Cabane, Bernard

2013-03-06

The skin is a barrier membrane that separates environments with profoundly different water contents. The barrier properties are assured by the outer layer of the skin, the stratum corneum (SC), which controls the transepidermal water loss. The SC acts as a responding membrane, since its hydration and permeability vary with the boundary condition, which is the activity of water at the outer surface of the skin. We show how this boundary condition can be changed by the application of a barrier cream that makes a film with a high resistance to the transport of water. We present a quantitative model that predicts hydration and water transport in SC that is covered by such a film. We also develop an experimental method for measuring the specific resistance to water transport of films made of occluding barrier creams. Finally, we combine the theoretical model with the measured properties of the barrier creams to predict how a film of cream changes the activity of water at the outer surface of the SC. Using the known variations of SC permeability and hydration with the water activity in its environment (i.e. the relative humidity), we can thus predict how a film of barrier cream changes SC hydration.

Mechanical stress regulates transport in a compliant 3D model of the blood-brain barrier.

PubMed

Partyka, Paul P; Godsey, George A; Galie, John R; Kosciuk, Mary C; Acharya, Nimish K; Nagele, Robert G; Galie, Peter A

2017-01-01

Transport of fluid and solutes is tightly controlled within the brain, where vasculature exhibits a blood-brain barrier and there is no organized lymphatic network facilitating waste transport from the interstitial space. Here, using a compliant, three-dimensional co-culture model of the blood-brain barrier, we show that mechanical stimuli exerted by blood flow mediate both the permeability of the endothelial barrier and waste transport along the basement membrane. Application of both shear stress and cyclic strain facilitates tight junction formation in the endothelial monolayer, with and without the presence of astrocyte endfeet in the surrounding matrix. We use both dextran perfusion and TEER measurements to assess the initiation and maintenance of the endothelial barrier, and microparticle image velocimetry to characterize the fluid dynamics within the in vitro vessels. Application of pulsatile flow to the in vitro vessels induces pulsatile strain to the vascular wall, providing an opportunity to investigate stretch-induced transport along the basement membrane. We find that a pulsatile wave speed of approximately 1 mm/s with Womersley number of 0.004 facilitates retrograde transport of high molecular weight dextran along the basement membrane between the basal endothelium and surrounding astrocytes. Together, these findings indicate that the mechanical stress exerted by blood flow is an important regulator of transport both across and along the walls of cerebral microvasculature. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of cell membranes in the regulation of lignification in pine cells

NASA Technical Reports Server (NTRS)

Hendrix, D. L.

1978-01-01

The identity of pine cell membranes bearing PAL enzyme activity, the isolation of a plasma membrane preparation from pine cells for testing as a regulatory barrier in lignification, and the measurement of the geopotential effect in pine stems are presented. A model to describe and predict the interaction of gravity and lignification of higher plants was developed.

Cholesterol effect on water permeability through DPPC and PSM lipid bilayers: a molecular dynamics study.

PubMed

Saito, Hiroaki; Shinoda, Wataru

2011-12-29

Water permeability of two different lipid bilayers of dipalmitoylphosphatidylcholine (DPPC) and palmitoylsphingomyelin (PSM) in the absence and presence of cholesterol (0-50 mol %) have been studied by molecular dynamics simulations to elucidate the molecular mechanism of the reduction in water leakage across the membranes by the addition of cholesterol. An enhanced free energy barrier was observed in these membranes with increased cholesterol concentration, and this was explained by the reduced cavity density around the cholesterol in the hydrophobic membrane core. There was an increase of trans conformers in the hydrophobic lipid chains adjacent to the cholesterol, which reduced the cavity density. The enhanced free energy barrier was found to be the main reason to reduce the water permeability with increased cholesterol concentration. At low cholesterol concentrations the PSM bilayer exhibited a higher free energy barrier than the DPPC bilayer for water permeation, while at greater than 30 mol % of cholesterol the difference became minor. This tendency for the PSM and DPPC bilayers to resemble each other at higher cholesterol concentrations was similar to commonly observed trends in several structural properties, such as order parameters, cross-sectional area per molecule, and cavity density profiles in the hydrophobic regions of bilayer membranes. These results demonstrate that DPPC and PSM bilayers with high cholesterol contents possess similar physical properties, which suggests that the solubility of cholesterol in these lipid bilayers has importance for an understanding of multicomponent lipid membranes with cholesterol. © 2011 American Chemical Society

Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway

PubMed Central

Caraballo, Juan Carlos; Yshii, Cecilia; Butti, Maria L.; Westphal, Whitney; Borcherding, Jennifer A.; Allamargot, Chantal

2011-01-01

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (Gt), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po2 = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased Gt. Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H2O2). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced Gt increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H2O2 during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in Gt. In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in Gt and occludin reduction at the plasma membrane in AEC. PMID:21257729

Debris buster is a Drosophila scavenger receptor essential for airway physiology.

PubMed

Wingen, Almut; Carrera, Pilar; Ekaterini Psathaki, Olympia; Voelzmann, André; Paululat, Achim; Hoch, Michael

2017-10-01

Scavenger receptors class B (SR-B) are multifunctional transmembrane proteins, which in vertebrates participate in lipid transport, pathogen clearance, lysosomal delivery and intracellular sorting. Drosophila has 14 SR-B members whose functions are still largely unknown. Here, we reveal a novel role for the SR-B family member Debris buster (Dsb) in Drosophila airway physiology. Larvae lacking dsb show yeast avoidance behavior, hypoxia, and severe growth defects associated with impaired elongation and integrity along the airways. Furthermore, in dsb mutant embryos, the barrier function of the posterior spiracles, which are critical for gas exchange, is not properly established and liquid clearance is locally impaired at the spiracular lumen. We found that Dsb is specifically expressed in a group of distal epithelial cells of the posterior spiracle organ and not throughout the entire airways. Furthermore, tissue-specific knockdown and rescue experiments demonstrate that Dsb function in the airways is only required in the posterior spiracles. Dsb localizes in intracellular vesicles, and a subset of these associate with lysosomes. However, we found that depletion of proteins involved in vesicular transport to the apical membrane, but not in lysosomal function, causes dsb-like airway elongation defects. We propose a model in which Dsb sorts components of the apical extracellular matrix which are essential for airway physiology. Since SR-B LIMP2-deficient mice show reduced expression of several apical plasma membrane proteins, sorting of proteins to the apical membrane is likely an evolutionary conserved function of Dsb and LIMP2. Our data provide insights into a spatially confined function of the SR-B Dsb in intracellular trafficking critical for the physiology of the whole tubular airway network. Copyright © 2017 Elsevier Inc. All rights reserved.

Heterozygous Null Bone Morphogenetic Protein Receptor Type 2 Mutations Promote SRC Kinase-dependent Caveolar Trafficking Defects and Endothelial Dysfunction in Pulmonary Arterial Hypertension*

PubMed Central

Prewitt, Allison R.; Ghose, Sampa; Frump, Andrea L.; Datta, Arumima; Austin, Eric D.; Kenworthy, Anne K.; de Caestecker, Mark P.

2015-01-01

Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2+/−) similar to those found in the majority of HPAH patients. We show that Bmpr2+/− PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2+/− PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2+/− PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2+/− PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients. PMID:25411245

GLUT-1 GLUCOSE TRANSPORTERS IN THE BLOOD-BRAIN BARRIER: DIFFERENTIAL PHOSPHORYLATION

PubMed Central

Devraj, Kavi; Klinger, Marianne E.; Myers, Roland L.; Mokashi, Ashwini; Hawkins, Richard A.; Simpson, Ian A.

2013-01-01

Glucose is the primary metabolic fuel for the mammalian brain and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood-brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT-1. Prior studies (Simpson et al. 2001) had revealed that the conformations of the GLUT-1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. In this study we have extended these observations and using a combination of 2D-PAGE/Western blotting and immunogold electron microscopy we determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT-1 and not from alternative splicing or altered O- or N-linked glycosylation. PMID:21910135

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

PubMed Central

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality. PMID:24505408

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

PubMed

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

Gold Nanoparticles-Enhanced Proton Exchange Membrane (PEM) Fuel Cell

NASA Astrophysics Data System (ADS)

Li, Hongfei; Pan, Cheng; Liu, Ping; Zhu, Yimei; Adzic, Radoslav; Rafailovich, Miriam

Proton exchange membrane fuel cells have drawn great attention and been taken as a promising alternated energy source. One of the reasons hamper the wider application of PEM fuel cell is the catalytic poison effect from the impurity of the gas flow. Haruta has predicted that gold nanoparticles that are platelet shaped and have direct contact with the metal oxide substrate to be the perfect catalysts of the CO oxidization, yet the synthesis method is difficult to apply in the Fuel Cell. In our approach, thiol-functionalized gold nanoparticles were synthesized through two-phase method developed by Brust et al. We deposit these Au particles with stepped surface directly onto the Nafion membrane in the PEM fuel cell by Langmuir-Blodgett method, resulting in over 50% enhancement of the efficiency of the fuel cell. DFT calculations were conducted to understand the theory of this kind of enhancement. The results indicated that only when the particles were in direct surface contact with the membrane, where AuNPs attached at the end of the Nafion side chains, it could reduce the energy barrier for the CO oxidation that could happen at T<300K.

Lamellar ichthyosis in a female neonate without a collodion membrane.

PubMed

Chao, Kevin; Aleshin, Maria; Goldstein, Zachary; Worswick, Scott; Hogeling, Marcia

2018-02-15

The term, autosomal recessive congenital ichthyosis (ARCI), describes a group of rare genetic skin diseases of cornification involving hyperkeratotic scaling at birth. The defective skin barrier function may lead to dehydration, body temperature instability, and high susceptibility to infections. In most cases of ARCI, neonates are born with a collodion membrane covering the body, often presenting with ectropion and eclabium. We report a premature female neonate presenting with hyperkeratotic scaling at birth without a collodion membrane. She was managed with placement in a humidified isolette, prophylactic antibiotics, dilute bleach baths, petrolatum ointment, and artificial eye drops. By the fourth week of life, there was marked improvement in her skin with the large, brown, plate-like scales on the trunk and extremities becoming lighter in color and finer in appearance. The ichthyosis genetic panel showed mutations in the ABCA12 gene resulting in the lamellar ichthyosis phenotype of ARCI. Our literature review revealed at least 28 patients with ARCI who were not born as collodion babies. Although collodion babies are a hallmark of most ARCI cases, clinicians should be aware of neonates with ARCI born without a collodion membrane and expedite appropriate workup and treatment.

Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence.

PubMed

Brown, Robert W B; Sharma, Aabha I; Engman, David M

2017-04-01

Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include - targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites - Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia - have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium.

PubMed

Fu, Xiang-Yang

2010-09-01

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.

Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease.

PubMed

Mak, Ki M; Mei, Rena

2017-08-01

Basement membranes provide structural support to epithelium, endothelium, muscles, fat cells, Schwann cells, and axons. Basement membranes are multifunctional: they modulate cellular behavior, regulate organogenesis, promote tissue repair, form a barrier to filtration and tumor metastasis, bind growth factors, and mediate angiogenesis. All basement membranes contain type IV collagen (Col IV), laminin, nidogen, and perlecan. Col IV and laminin self-assemble into two independent supramolecular networks that are linked to nidogen and perlecan to form a morphological discernable basement membrane/basal lamina. The triple helical region, 7S domain and NCI domain of Col IV, laminin and laminin fragment P1 have been evaluated as noninvasive fibrosis biomarkers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. Elevated serum Col IV and laminin are related to degrees of fibrosis and severity of hepatitis, and may reflect hepatic basement membrane metabolism. But the serum assays have not been linked to disclosing the anatomical sites and lobular distribution of perisinusoidal basement membrane formation in the liver. Hepatic sinusoids normally lack a basement membrane, although Col IV is a normal matrix component of the space of Disse. In liver disease, laminin deposits in the space of Disse and codistributes with Col IV, forming a perisinusoidal basement membrane. Concomitantly, the sinusoidal endothelium loses its fenestrae and is transformed into vascular type endothelium. These changes lead to capillarization of hepatic sinusoids, a significant pathology that impairs hepatic function. Accordingly, codistribution of Col IV and laminin serves as histochemical marker of perisinusoidal basement membrane formation in liver disease. Anat Rec, 300:1371-1390, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Transport of Zn(OH)4(-2) ions across a polyolefin microporous membrane

NASA Astrophysics Data System (ADS)

Krejci, Ivan; Vanysek, Peter; Trojanek, Antonin

1993-04-01

Transport of ZN(OH)4(2-) ions through modified microporous polypropylene membranes (Celgard 3401, 350140) was studied using polarography and conductometry. Soluble Nafion as an ion exchange modifying agent was applied to the membrane by several techniques. The influence of Nafion and a surfactant on transport of zinc ions through the membrane was studied. A relationship between membrane impedance and the rate of Zn(OH)4(2-) transport was found. The found correlation between conductivity, ion permeability and Nafion coverage suggests a suitable technique of membrane preparation to obtain desired zinc ion barrier properties.

Profound re-organization of cell surface proteome in equine retinal pigment epithelial cells in response to in vitro culturing.

PubMed

Szober, Christoph M; Hauck, Stefanie M; Euler, Kerstin N; Fröhlich, Kristina J H; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A

2012-10-31

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

Development of an implantable synthetic membrane for the treatment of preterm premature rupture of fetal membranes.

PubMed

Roman, Sabiniano; Bullock, Anthony J; Anumba, Dilly O; MacNeil, Sheila

2016-02-01

Preterm premature rupture of fetal membranes is a very common condition leading to premature labour of a non viable fetus. Significant morbidities may occur when preterm premature rupture of fetal membranes management is attempted to prolong the pregnancy for fetal maturation. Reducing the rate of loss of amniotic fluid and providing a barrier to bacterial entry may allow the pregnancy to continue to term, avoiding complications. Our aim is to develop a synthetic biocompatible membrane to form a distensible barrier for cervical closure which acts to reduce fluid loss and provide a surface for epithelial ingrowth to help repair the damaged membranes. Therefore, a bilayer membrane was developed using an electrospinning technique of combining two FDA-approved polymers, poly-L-lactic acid (PLA) and polyurethane (Z3) polymer. This was compared to a plain electrospun Z3 membrane. The physical and mechanical properties were assessed using scanning electron microscope images and a BOSE tensiometer, respectively, and compared to native fetal membranes. The performance of the membranes in preventing fluid loss was assessed by measuring their ability to support a column of water. Finally the ability of the membranes to support cell ingrowth was assessed by culturing adipose-derived stem cells on the membranes for two weeks and assessing metabolic activity after 7 and 14 days. The physical properties of the bilayer were similar to that of the native fetal membranes and it was resistant to fluid penetration. This bilayer membrane presented mechanical properties close to those for fetal membranes and showed elastic distention, which may be crucial for progress of the pregnancy. The membrane was also able to retain surgical sutures. In addition, it also supported the attachment and growth of adipose-derived stem cells for two weeks. In conclusion, this membrane may prove a useful approach in the treatment of preterm premature rupture of fetal membranes and now merits further investigation. © The Author(s) 2015.

Lipid rafts are disrupted in mildly inflamed intestinal microenvironments without overt disruption of the epithelial barrier.

PubMed

Bowie, Rachel V; Donatello, Simona; Lyes, Clíona; Owens, Mark B; Babina, Irina S; Hudson, Lance; Walsh, Shaun V; O'Donoghue, Diarmuid P; Amu, Sylvie; Barry, Sean P; Fallon, Padraic G; Hopkins, Ann M

2012-04-15

Intestinal epithelial barrier disruption is a feature of inflammatory bowel disease (IBD), but whether barrier disruption precedes or merely accompanies inflammation remains controversial. Tight junction (TJ) adhesion complexes control epithelial barrier integrity. Since some TJ proteins reside in cholesterol-enriched regions of the cell membrane termed lipid rafts, we sought to elucidate the relationship between rafts and intestinal epithelial barrier function. Lipid rafts were isolated from Caco-2 intestinal epithelial cells primed with the proinflammatory cytokine interferon-γ (IFN-γ) or treated with methyl-β-cyclodextrin as a positive control for raft disruption. Rafts were also isolated from the ilea of mice in which colitis had been induced in conjunction with in vivo intestinal permeability measurements, and lastly from intestinal biopsies of ulcerative colitis (UC) patients with predominantly mild or quiescent disease. Raft distribution was analyzed by measuring activity of the raft-associated enzyme alkaline phosphatase and by performing Western blot analysis for flotillin-1. Epithelial barrier integrity was estimated by measuring transepithelial resistance in cytokine-treated cells or in vivo permeability to fluorescent dextran in colitic mice. Raft and nonraft fractions were analyzed by Western blotting for the TJ proteins occludin and zonula occludens-1 (ZO-1). Our results revealed that lipid rafts were disrupted in IFN-γ-treated cells, in the ilea of mice with subclinical colitis, and in UC patients with quiescent inflammation. This was not associated with a clear pattern of occludin or ZO-1 relocalization from raft to nonraft fractions. Significantly, a time-course study in colitic mice revealed that disruption of lipid rafts preceded the onset of increased intestinal permeability. Our data suggest for the first time that lipid raft disruption occurs early in the inflammatory cascade in murine and human colitis and, we speculate, may contribute to subsequent disruption of epithelial barrier function.

Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development.

PubMed

Wong, Bernice H; Chan, Jia Pei; Cazenave-Gassiot, Amaury; Poh, Rebecca W; Foo, Juat Chin; Galam, Dwight L A; Ghosh, Sujoy; Nguyen, Long N; Barathi, Veluchamy A; Yeo, Sia W; Luu, Chi D; Wenk, Markus R; Silver, David L

2016-05-13

Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation.

PubMed

Nasseau, M; Boublik, Y; Meier, W; Winterhalter, M; Fournier, D

2001-12-05

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. Copyright 2001 John Wiley & Sons, Inc.

Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins

PubMed Central

Wong, Elissa WP; Lie, Pearl PY; Li, Michelle WM; Mruk, Dolores D; Yan, Helen HN; Mok, Ka-Wai; Mannu, Jayakanthan; Mathur, Premendu P; Lui, Wing-yee; Lee, Will M; Bonanomi, Michele; Silvestrini, Bruno

2011-01-01

The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the “gateway” to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction. PMID:22319658

The biological significance of brain barrier mechanisms: help or hindrance in drug delivery to the central nervous system?

PubMed Central

Saunders, Norman R.; Habgood, Mark D.; Møllgård, Kjeld; Dziegielewska, Katarzyna M.

2016-01-01

Barrier mechanisms in the brain are important for its normal functioning and development. Stability of the brain’s internal environment, particularly with respect to its ionic composition, is a prerequisite for the fundamental basis of its function, namely transmission of nerve impulses. In addition, the appropriate and controlled supply of a wide range of nutrients such as glucose, amino acids, monocarboxylates, and vitamins is also essential for normal development and function. These are all cellular functions across the interfaces that separate the brain from the rest of the internal environment of the body. An essential morphological component of all but one of the barriers is the presence of specialized intercellular tight junctions between the cells comprising the interface: endothelial cells in the blood-brain barrier itself, cells of the arachnoid membrane, choroid plexus epithelial cells, and tanycytes (specialized glial cells) in the circumventricular organs. In the ependyma lining the cerebral ventricles in the adult brain, the cells are joined by gap junctions, which are not restrictive for intercellular movement of molecules. But in the developing brain, the forerunners of these cells form the neuroepithelium, which restricts exchange of all but the smallest molecules between cerebrospinal fluid and brain interstitial fluid because of the presence of strap junctions between the cells. The intercellular junctions in all these interfaces are the physical basis for their barrier properties. In the blood-brain barrier proper, this is combined with a paucity of vesicular transport that is a characteristic of other vascular beds. Without such a diffusional restrain, the cellular transport mechanisms in the barrier interfaces would be ineffective. Superimposed on these physical structures are physiological mechanisms as the cells of the interfaces contain various metabolic transporters and efflux pumps, often ATP-binding cassette (ABC) transporters, that provide an important component of the barrier functions by either preventing entry of or expelling numerous molecules including toxins, drugs, and other xenobiotics. In this review, we summarize these influx and efflux mechanisms in normal developing and adult brain, as well as indicating their likely involvement in a wide range of neuropathologies. There have been extensive attempts to overcome the barrier mechanisms that prevent the entry of many drugs of therapeutic potential into the brain. We outline those that have been tried and discuss why they may so far have been largely unsuccessful. Currently, a promising approach appears to be focal, reversible disruption of the blood-brain barrier using focused ultrasound, but more work is required to evaluate the method before it can be tried in patients. Overall, our view is that much more fundamental knowledge of barrier mechanisms and development of new experimental methods will be required before drug targeting to the brain is likely to be a successful endeavor. In addition, such studies, if applied to brain pathologies such as stroke, trauma, or multiple sclerosis, will aid in defining the contribution of brain barrier pathology to these conditions, either causative or secondary. PMID:26998242

Conception and construction of an LPG tank using a composite membrane technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fuvel, P.; Claude, J.

TECHNIGAZ and TOTAL C.F.P. have developed a new LPG storage technology derived from the membrane concept used for LNG storage and transportation. This technology called GMS uses a composite membrane as primary barrier. A 2 000 m/sup 3/ storage pilot unit, based on that concept, is under construction in TOTAL's refinery at DUNKIRK (France) since September 1983.

LOCATING AND REPAIRING LEAKS IN LANDFILL/IMPOUNDMENT FLEXIBLE MEMBRANE LINERS

EPA Science Inventory

In the United States, the large quantities of solid and hazardous wastes generated each year are commonly disposed of in landfills and surface impoundments. Geomembrane liners (flexible membrane liners, FMLs) are often used to form an impermeable barrier to prevent migration of c...

Insight of Transmembrane Processes of Self-Assembling Nanotubes Based on a Cyclic Peptide Using Coarse Grained Molecular Dynamics Simulation.

PubMed

Fu, Yankai; Yan, Tingxuan; Xu, Xia

2017-09-28

Transmembrane self-assembling cyclic peptide (SCP) nanotubes are promising candidates for delivering specific molecules through cell membranes. The detailed mechanisms behind the transmembrane processes, as well as stabilization factors of transmembrane structures, are difficult to elucidate through experiments. In this study, the effects of peptide sequence and oligomeric state on the transmembrane capabilities of SCP nanotubes and the perturbation of embedded SCP nanotubes acting on the membrane were investigated based on coarse grained molecular dynamics simulation. The simulation results reveal that hydrophilic SCP oligomers result in the elevation of the energy barrier while the oligomerization of hydrophobic SCPs causes the reduction of the energy barrier, further leading to membrane insertion. Once SCP nanotubes are embedded, membrane properties such as density, thickness, ordering state and lateral mobility are adjusted along the radial direction. This study provides insight into the transmembrane strategy of SCP nanotubes and sheds light on designing novel transport systems.

The unwound portion dividing helix IV of NhaA undergoes a conformational change at physiological pH and lines the cation passage.

PubMed

Rimon, Abraham; Kozachkov-Magrisso, Lena; Padan, Etana

2012-11-27

pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and their pH regulation. Functional studies of NhaA in the membrane have yielded valuable information regarding its functionality in situ at physiological pH. Here, we Cys-scanned the discontinuous transmembrane segment (TM) IV (helices IVp and IVc connected by an extended chain) of NhaA to explore its functionality at physiological pH. We then tested the accessibility of the Cys replacements to the positively charged SH reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and the negatively charged 2-sulfonatoethyl methanethiosulfonate (MTSES) in intact cells at pH 8.5 and 6.5 and in parallel tested their accessibility to MTSET in high-pressure membranes at both pH values. We found that the outer membrane of E. coli TA16 acts as a partially permeable barrier to MTSET. Overcoming this technical problem, we revealed that (a) Cys replacement of the most conserved residues of TM IV strongly increases the apparent K(m) of NhaA to both Na(+) and Li(+), (b) the cationic passage of NhaA at physiological pH is lined by the most conserved and functionally important residues of TM IV, and (c) a pH shift from 6.5 to 8.5 induces conformational changes in helix IVp and in the extended chain at physiological pH.

Putative role of membranes in the HIV fusion inhibitor enfuvirtide mode of action at the molecular level.

PubMed Central

Veiga, Salomé; Henriques, Sónia; Santos, Nuno C; Castanho, Miguel

2004-01-01

Partition of the intrinsically fluorescent HIV fusion inhibitor enfuvirtide into lipidic membranes is relatively high (Delta G =6.6 kcal x mol(-1)) and modulated by cholesterol. A shallow position in the lipidic matrix makes it readily available for interaction with gp41. No conformational energetic barrier prevents enfuvirtide from being active in both aqueous solution and lipidic membranes. Lipidic membranes may play a key role in the enfuvirtide biochemical mode of action. PMID:14514352

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

PubMed

Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

2016-03-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

PubMed Central

Min, Kyoung Ah; Rosania, Gus R.; Kim, Chong-Kook; Shin, Meong Cheol

2016-01-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies. PMID:26746641

Cytokine Signaling Modulates Blood-Brain Barrier Function

PubMed Central

Pan, Weihong; Stone, Kirsten P.; Hsuchou, Hung; Manda, Vamshi K.; Zhang, Yan; Kastin, Abba J.

2014-01-01

The blood-brain barrier (BBB) provides a vast interface for cytokines to affect CNS function. The BBB is a target for therapeutic intervention. It is essential, therefore, to understand how cytokines interact with each other at the level of the BBB and how secondary signals modulate CNS functions beyond the BBB. The interactions between cytokines and lipids, however, have not been fully addressed at the level of the BBB. Here, we summarize current understanding of the localization of cytokine receptors and transporters in specific membrane microdomains, particularly lipid rafts, on the luminal (apical) surface of the microvascular endothelial cells composing the BBB. We then illustrate the clinical context of cytokine effects on the BBB by neuroendocrine regulation and amplification of inflammatory signals. Two unusual aspects discussed are signaling crosstalk by different classes of cytokines and genetic regulation of drug efflux transporters. We also introduce a novel area of focus on how cytokines may act through nuclear hormone receptors to modulate efflux transporters and other targets. A specific example discussed is the ATP-binding cassette transporter-1 (ABCA-1) that regulates lipid metabolism. Overall, cytokine signaling at the level of the BBB is a crucial feature of the dynamic regulation that can rapidly change BBB function and affect brain health and disease. PMID:21834767

Fatty Aldehyde and Fatty Alcohol Metabolism: Review and Importance for Epidermal Structure and Function

PubMed Central

Rizzo, William B.

2014-01-01

Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren-Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. PMID:24036493

HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

PubMed Central

García-Expósito, Laura; Barroso-González, Jonathan; Puigdomènech, Isabel; Machado, José-David; Blanco, Julià; Valenzuela-Fernández, Agustín

2011-01-01

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4+ T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4+ T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry. PMID:21346189

Effect of heterogeneity on the characterization of cell membrane compartments: I. Uniform size and permeability.

PubMed

Hall, Damien

2010-03-15

Observations of the motion of individual molecules in the membrane of a number of different cell types have led to the suggestion that the outer membrane of many eukaryotic cells may be effectively partitioned into microdomains. A major cause of this suggested partitioning is believed to be due to the direct/indirect association of the cytosolic face of the cell membrane with the cortical cytoskeleton. Such intimate association is thought to introduce effective hydrodynamic barriers into the membrane that are capable of frustrating molecular Brownian motion over distance scales greater than the average size of the compartment. To date, the standard analytical method for deducing compartment characteristics has relied on observing the random walk behavior of a labeled lipid or protein at various temporal frequencies and different total lengths of time. Simple theoretical arguments suggest that the presence of restrictive barriers imparts a characteristic turnover to a plot of mean squared displacement versus sampling period that can be interpreted to yield the average dimensions of the compartment expressed as the respective side lengths of a rectangle. In the following series of articles, we used computer simulation methods to investigate how well the conventional analytical strategy coped with heterogeneity in size, shape, and barrier permeability of the cell membrane compartments. We also explored questions relating to the necessary extent of sampling required (with regard to both the recorded time of a single trajectory and the number of trajectories included in the measurement bin) for faithful representation of the actual distribution of compartment sizes found using the SPT technique. In the current investigation, we turned our attention to the analytical characterization of diffusion through cell membrane compartments having both a uniform size and permeability. For this ideal case, we found that (i) an optimum sampling time interval existed for the analysis and (ii) the total length of time for which a trajectory was recorded was a key factor. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Modulation of N-glycosylation by mesalamine facilitates membranous E-cadherin expression in colon epithelial cells☆

PubMed Central

Khare, Vineeta; Lang, Michaela; Dammann, Kyle; Campregher, Christoph; Lyakhovich, Alex; Gasche, Christoph

2014-01-01

Genome wide association studies have implicated intestinal barrier function genes in the pathogenesis of ulcerative colitis. One of such loci CDH1, encoding E-cadherin, a transmembrane glycoprotein with known tumor suppressor functions, is also linked to the susceptibility to colorectal cancer. Loss of membranous E-cadherin expression is common in both colitis and cancer. We have recently demonstrated that mesalamine (5-ASA); the anti-inflammatory drug used to treat ulcerative colitis, induces membranous expression of E-cadherin and increases intercellular adhesion. Using colorectal cancer epithelial cells with aberrant E-cadherin expression, we investigated the mechanism underlying such an effect of 5-ASA. Post-translational modification of E-cadherin glycosylation was analyzed by biotin/streptavidin detection of sialylated glycoproteins. GnT-III (N-acetylglucosaminyltransferase III) expression was assessed by qRT-PCR, Western blot and immunofluorescence. GnT-III activity was analyzed by reactivity with E-4/L-4-PHA. Expression, localization and interaction of E-cadherin and β-catenin were analyzed by Western blot, immunocytochemistry and RNA interference. 5-ASA activity modulated E-cadherin glycosylation and increased both mRNA and protein levels of GnT-III and its activity as detected by increased E4-lectin reactivity. Intestinal APCMin polyps in mice showed low expression of GnT-III and 5-ASA was effective in increasing its expression. The data demonstrated that remodeling of glycans by GnT-III mediated bisect glycosylation, contributes to the membranous retention of E-cadherin by 5-ASA; facilitating intercellular adhesion. Induction of membranous expression of E-cadherin by 5-ASA is a novel mechanism for mucosal healing in colitis that might impede tumor progression by modulation of GnT-III expression. PMID:24184502

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

PubMed Central

Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

2016-01-01

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

EMMPRIN modulates epithelial barrier function through a MMP-mediated occludin cleavage: implications in dry eye disease.

PubMed

Huet, Eric; Vallée, Benoit; Delbé, Jean; Mourah, Samia; Prulière-Escabasse, Virginie; Tremouilleres, Magali; Kadomatsu, Kenji; Doan, Serge; Baudouin, Christophe; Menashi, Suzanne; Gabison, Eric E

2011-09-01

Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Efficient hydrogen isotopologues separation through a tunable potential barrier: The case of a C2N membrane.

PubMed

Qu, Yuanyuan; Li, Feng; Zhao, Mingwen

2017-05-03

Isotopes separation through quantum sieving effect of membranes is quite promising for industrial applications. For the light hydrogen isotopologues (eg. H 2 , D 2 ), the confinement of potential wells in porous membranes to isotopologues was commonly regarded to be crucial for highly efficient separation ability. Here, we demonstrate from first-principles that a potential barrier is also favorable for efficient hydrogen isotopologues separation. Taking an already-synthesized two-dimensional carbon nitride (C 2 N-h2D) as an example, we predict that the competition between quantum tunneling and zero-point-energy (ZPE) effects regulated by the tensile strain leads to high selectivity and permeance. Both kinetic quantum sieving and equilibrium quantum sieving effects are considered. The quantum effects revealed in this work offer a prospective strategy for highly efficient hydrogen isotopologues separation.

Conductivity equations of protons transporting through 2D crystals obtained with the rate process theory and free volume concept

NASA Astrophysics Data System (ADS)

Hao, Tian; Xu, Yuanze; Hao, Ting

2018-04-01

The Eyring's rate process theory and free volume concept are employed to treat protons (or other particles) transporting through a 2D (two dimensional) crystal like graphene and hexagonal boron nitride. The protons are assumed to be activated first in order to participate conduction and the conduction rate is dependent on how much free volume available in the system. The obtained proton conductivity equations show that only the number of conduction protons, proton size and packing structure, and the energy barrier associated with 2D crystals are critical; the quantization conductance is unexpectedly predicted with a simple Arrhenius type temperature dependence. The predictions agree well with experimental observations and clear out many puzzles like much smaller energy barrier determined from experiments than from the density function calculations and isotope separation rate independent of the energy barrier of 2D crystals, etc. Our work may deepen our understandings on how protons transport through a membrane and has direct implications on hydrogen related technology and proton involved bioprocesses.

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

PubMed Central

Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

2015-01-01

The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

A nonsurgical endodontic treatment in open-apex and immature teeth affected by dens invaginatus: using a collagen membrane as an apical barrier.

PubMed

Gharechahi, Maryam; Ghoddusi, Jamileh

2012-02-01

The authors' objective in this case report is to demonstrate an effective nonsurgical endodontic treatment in open-apex teeth affected by dens invaginatus (DI) by using a collagen membrane as an apical barrier and using a mineral trioxide aggregate (MTA) apical plug. . The authors present two cases of DI with open apexes in maxillary lateral incisors. In the first case, an adolescent had bilateral Oehlers type II DI and extensive periradicular radiolucency, internal root resorption and a vestibular fistula in the left maxillary lateral incisor. In the second case, an adult had Oehlers type II DI and an incomplete apex in the left maxillary lateral incisor. For both patients, the clinician placed a collagen membrane through the apexes of the left maxillary incisors to provide a resorbable extraradicular barrier against which MTA cement could be packed. The clinician obturated the adolescent's right lateral incisor. In the adolescent, the vestibular sinus tract was closed after one week. At subsequent follow-up examinations, the periradicular regions were completely healed, and postoperative radiographs revealed good bone healing in the lateral incisors. The teeth were asymptomatic and healing was achieved without any need for further endodontic surgical intervention. In the adult patient, the tooth was symptom free after one week, and radiography performed six months after the procedure showed complete healing. and Despite complex anatomy and diagnoses of DI and open apexes, both patients successfully underwent nonsurgical endodontic treatment involving the use of a collagen membrane and an MTA apical plug. Using an extraradicular barrier clinically can help improve the adaptation of MTA in the apexes of open-apex teeth to achieve a complete seal.

Effects of autoclave sterilization on properties of dental rubber dam as related to its use as barrier membrane in guided tissue regeneration.

PubMed

Apinhasmit, Wandee; Limsombutanon, Somchai; Swasdison, Somporn; Suppipat, Nophadol

2003-10-01

Dental rubber dams (RDs) were used as barrier membranes in guided tissue regeneration for the treatment of periodontal intraosseous defects with acceptable clinical results. The aim of the present study was to investigate the effects of autoclave sterilization on properties of RD as related to its use as a barrier membrane in guided tissue regeneration. RDs were sterilized by either an autoclave, gamma irradiation, or chemical agents and then co-cultured with human gingival fibroblasts. The cell responses to sterilized RDs were investigated by inverted phase contrast microscopy, scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) technique. The surface alterations of the autoclaved RDs were observed under SEM. The tensile strength, tear strength and elongation at break of the autoclaved RDs were tested by a universal testing machine. The results from cell culture, microscopic and MTT studies showed that RDs sterilized by autoclave and gamma irradiation did not deteriorate gingival fibroblasts and provided surfaces suitable for cell attachment, whereas chemical-sterilized RDs were toxic to these cells. Ultrastructurally, surface changes from the non-autoclaved RDs, including some melted areas, small pores and folds were observed on the autoclaved RD surface. The tensile strength and tear strength of the autoclaved RDs were significantly lower than those of the non-autoclaved RDs (p = 0.042, p < 0.001, respectively). In contrast, the elongation at break of the autoclaved RDs was higher than that of the non-autoclaved RDs (p < 0.001). These results suggest that the autoclave sterilization deteriorated the physical properties of RDs even though they seemed to be compatible to the cultured human cells. Therefore, the sterilization method should be taken into consideration when RDs are utilized as barrier membranes.

Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli.

PubMed

Bushell, Simon R; Mainprize, Iain L; Wear, Martin A; Lou, Hubing; Whitfield, Chris; Naismith, James H

2013-05-07

Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides. Copyright © 2013 Elsevier Ltd. All rights reserved.

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

PubMed

Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

2015-08-01

Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Rac-specific exchange factors Dock1 and Dock5 are dispensable for the establishment of the glomerular filtration barrier in vivo

PubMed Central

Laurin, Mélanie; Dumouchel, Annie; Fukui, Yoshinori; Côté, Jean-François

2013-01-01

Podocytes are specialized kidney cells that form the kidney filtration barrier through the connection of their foot processes. Nephrin and Neph family transmembrane molecules at the surface of podocytes interconnect to form a unique type of cell-cell junction, the slit diaphragm, which acts as a molecular sieve. The cytoplasmic tails of Nephrin and Neph mediate cytoskeletal rearrangement that contributes to the maintenance of the filtration barrier. Nephrin and Neph1 orthologs are essential to regulate cell-cell adhesion and Rac-dependent actin rearrangement during Drosophila myoblast fusion. We hypothesized here that molecules regulating myoblast fusion in Drosophila could contribute to signaling downstream of Nephrin and Neph1 in podocytes. We found that Nephrin engagement promoted recruitment of the Rac exchange factor Dock1 to the membrane. Furthermore, Nephrin overexpression led to lamellipodia formation that could be blocked by inhibiting Rac1 activity. We generated in vivo mouse models to investigate whether Dock1 and Dock5 contribute to the formation and maintenance of the kidney filtration barrier. Our results indicate that while Dock1 and Dock5 are expressed in podocytes, their functions are not essential for the development of the glomerular filtration barrier. Furthermore, mice lacking Dock1 were not protected from LPS-induced podocyte effacement. Our data suggest that Dock1 and Dock5 are not the important exchange factors regulating Rac activity during the establishment and maintenance of the glomerular barrier. PMID:24365888

Guided Bone Regeneration in Long-Bone Defects with a Structural Hydroxyapatite Graft and Collagen Membrane

DTIC Science & Technology

2013-01-01

Praetorius, F. Guided tissue regeneration using de- gradable and nondegradable membranes in rabbit tibia. Clin Oral Implants Res 4, 172, 1993. 8. Queiroz... Regeneration of periodontal tissues : combinations of barrier membranes and grafting materials–biological foundation and preclinical evi- dence: a...structural graft provides benefits for bone tissue regeneration in terms of early interfacial integration. Introduction The treatment of large-bone defects

Theory and simulation of ion conduction in the pentameric GLIC channel.

PubMed

Zhu, Fangqiang; Hummer, Gerhard

2012-10-09

GLIC is a bacterial member of the large family of pentameric ligand-gated ion channels. To study ion conduction through GLIC and other membrane channels, we combine the one-dimensional potential of mean force for ion passage with a Smoluchowski diffusion model, making it possible to calculate single-channel conductance in the regime of low ion concentrations from all-atom molecular dynamics (MD) simulations. We then perform MD simulations to examine sodium ion conduction through the GLIC transmembrane pore in two systems with different bulk ion concentrations. The ion potentials of mean force, calculated from umbrella sampling simulations with Hamiltonian replica exchange, reveal a major barrier at the hydrophobic constriction of the pore. The relevance of this barrier for ion transport is confirmed by a committor function that rises sharply in the barrier region. From the free evolution of Na(+) ions starting at the barrier top, we estimate the effective diffusion coefficient in the barrier region, and subsequently calculate the conductance of the pore. The resulting diffusivity compares well with the position-dependent ion diffusion coefficient obtained from restrained simulations. The ion conductance obtained from the diffusion model agrees with the value determined via a reactive-flux rate calculation. Our results show that the conformation in the GLIC crystal structure, with an estimated conductance of ~1 picosiemens at 140 mM ion concentration, is consistent with a physiologically open state of the channel.

Graphite oxide incorporated crosslinked polyvinyl alcohol and sulfonated styrene nanocomposite membrane as separating barrier in single chambered microbial fuel cell

NASA Astrophysics Data System (ADS)

Rudra, Ruchira; Kumar, Vikash; Pramanik, Nilkamal; Kundu, Patit Paban

2017-02-01

Different membranes with varied molar concentrations of graphite oxide (GO), 'in situ' polymerized sulfonated polystyrene (SS) and glutaraldehyde (GA) cross linked polyvinyl alcohol (PVA), have been analyzed as an effective and low cost nanocomposite barrier in single chambered microbial fuel cells (MFCs). The synthesized composite membranes, namely GO0.2, GO0.4 and GO0.6 exhibited comparatively better results with reduced water uptake (WU) and swelling ratios (SR) over the native PVA. The variation in properties is illustrated with membrane analyses, where GO0.4 showed an increased proton conductivity (PC) and ion exchange capacity (IEC) of 0.128 S cm-1 and 0.33 meq g-1 amongst all of the used membranes. In comparison, reduced oxygen diffusivity with lower water uptake showed a two-fold decrease in GO0.4 over pure PVA membrane (∼2.09 × 10-4 cm s-1). A maximum power density of 193.6 mW m-2 (773.33 mW m-3) with a current density of 803.33 mA m-2 were observed with GO0.4 fitted MFC, where ∼81.89% of chemical oxygen demand (COD) was removed using mixed firmicutes, as biocatalyst, in 25 days operation. In effect, the efficacy of GO incorporated crosslinked PVA and SS nanocomposite membrane has been evaluated as a polymer electrolyte membrane for harnessing bio-energy from single chambered MFCs.

Structure of a CLC chloride ion channel by cryo-electron microscopy

PubMed Central

Park, Eunyong; Campbell, Ernest B.; MacKinnon, Roderick

2017-01-01

CLC proteins transport chloride (Cl−) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl− ions passively, whereas others are secondary active transporters that exchange two Cl− ions for one H+. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture based on sequence homology. To solve this puzzle we determined the structure of a mammalian CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl− transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl− passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl−/H+ transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl− down its electrochemical gradient. PMID:28002411

Asymmetric distribution of charged lipids between the leaflets of a vesicle bilayer induced by melittin and alamethicin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Shuo; Heller, William T

2011-01-01

Cellular membranes are complex mixtures of lipids, proteins, and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. The membrane structure results from the complex interplay between the wide varieties of molecules present. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoylmore » phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than it is for alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state. The results suggest that these membrane-active peptides may have a secondary stressful effect on target cells at low concentrations that results from a disruption of the lipid distribution between the inner and outer leaflets of the bilayer that is independent of the formation of transmembrane pores.« less

The simulation approach to lipid-protein interactions.

PubMed

Paramo, Teresa; Garzón, Diana; Holdbrook, Daniel A; Khalid, Syma; Bond, Peter J

2013-01-01

The interactions between lipids and proteins are crucial for a range of biological processes, from the folding and stability of membrane proteins to signaling and metabolism facilitated by lipid-binding proteins. However, high-resolution structural details concerning functional lipid/protein interactions are scarce due to barriers in both experimental isolation of native lipid-bound complexes and subsequent biophysical characterization. The molecular dynamics (MD) simulation approach provides a means to complement available structural data, yielding dynamic, structural, and thermodynamic data for a protein embedded within a physiologically realistic, modelled lipid environment. In this chapter, we provide a guide to current methods for setting up and running simulations of membrane proteins and soluble, lipid-binding proteins, using standard atomistically detailed representations, as well as simplified, coarse-grained models. In addition, we outline recent studies that illustrate the power of the simulation approach in the context of biologically relevant lipid/protein interactions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzales, Ivana; Chung, Hoon Taek; Kim, Yu Seung

Slow hydrogen oxidation reaction (HOR) kinetics on Pt under alkaline conditions is a significant technical barrier for the development of high-performance hydroxide exchange membrane fuel cells. Here we report that benzene adsorption on Pt is a major factor responsible for the sluggish HOR. Furthermore, we demonstrate that bimetallic catalysts, such as PtMo/C, PtNi/C, and PtRu/C, can reduce the adsorption of benzene and thereby improve HOR activity. In particular, the HOR voltammogram of PtRu/C in 0.1 M benzyl ammonium showed minimal benzene adsorption. Density functional theory calculations indicate that the adsorption of benzyl ammonium on the bimetallic PtRu is endergonic formore » all four possible orientations of the cation, which explains the significantly better HOR activity observed for the bimetallic catalysts. In conclusion, the new HOR inhibition mechanism described here provides insights for the design of future polymer electrolytes and electrocatalysts for better-performing polymer membrane-based fuel cells.« less

Free energy barrier for melittin reorientation from a membrane-bound state to a transmembrane state.

PubMed

Irudayam, Sheeba J; Pobandt, Tobias; Berkowitz, Max L

2013-10-31

An important step in a phospholipid membrane pore formation by melittin antimicrobial peptide is a reorientation of the peptide from a surface into a transmembrane conformation. Experiments measure the fraction of peptides in the surface state and the transmembrane state, but no computational study exists that quantifies the free energy curve for the reorientation. In this work we perform umbrella sampling simulations to calculate the potential of mean force (PMF) for the reorientation of melittin from a surface-bound state to a transmembrane state and provide a molecular level insight in understanding the peptide-lipid properties that influence the existence of the free energy barrier. The PMFs were calculated for a peptide to lipid (P/L) ratio of 1/128 and 4/128. We observe that the free energy barrier is reduced when the P/L ratio increases. In addition, we study the cooperative effect; specifically we investigate if the reorientation barrier is smaller for a second melittin, given that another neighboring melittin was already in the transmembrane orientation. We observe that indeed the barrier of the PMF curve is reduced in this case, thus confirming the presence of a cooperative effect.

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains.

PubMed

Villa, Riccardo; Martorana, Alessandra M; Okuda, Suguru; Gourlay, Louise J; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel; Polissi, Alessandra

2013-03-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

PubMed Central

Villa, Riccardo; Martorana, Alessandra M.; Okuda, Suguru; Gourlay, Louise J.; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel

2013-01-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components. PMID:23292770

The role of "blebbing" in overcoming the hydrophobic barrier during biooxidation of elemental sulfur by Thiobacillus thiooxidans

USGS Publications Warehouse

Knickerbocker, C.; Nordstrom, D. Kirk; Southam, G.

2000-01-01

Brimstone Basin, in southeastern Yellowstone National Park, Wyoming is an ancient hydrothermal area containing solfataric alteration. Drainage waters flowing from Brimstone Basin had pH values as low as 1.23 and contained up to 1.7×106 MPN/ml acidophilic sulfur-oxidizing bacteria. Thiobacillus thiooxidans was the dominant sulfur-oxidizing bacterium recovered from an enrichment culture and was used in a structural examination of bacterial sulfur oxidation. Growth in these sulfur cultures occurred in two phases with cells in association with the macroscopic sulfur grains and in suspension above these grains. Colonization of sulfur grains by individual cells and microcolonies was facilitated by organic material that appeared to be responsible for bacterial adhesion. Transmission electron microscopy of negatively stained (2% [wt./vol.] uranyl acetate), sulfur-grown T. thiooxidans revealed extensive membrane blebbing (sloughing of outer membrane vesicles) and the presence of approximately 100 nm sized sulfur particles adsorbed to membrane material surrounding individual bacteria. Sulfite-grown bacteria did not possess membrane blebs. The amphipathic nature of these outer membrane vesicles appear to be responsible for overcoming the hydrophobic barrier necessary for the growth of T. thiooxidans on elemental sulfur.

Endothelial glycocalyx: permeability barrier and mechanosensor.

PubMed

Curry, F E; Adamson, R H

2012-04-01

Endothelial cells are covered with a polysaccharide rich layer more than 400 nm thick, mechanical properties of which limit access of circulating plasma components to endothelial cell membranes. The barrier properties of this endothelial surface layer are deduced from the rate of tracer penetration into the layer and the mechanics of red and white cell movement through capillary microvessels. This review compares the mechanosensor and permeability properties of an inner layer (100-150 nm, close to the endothelial membrane) characterized as a quasi-periodic structure which accounts for key aspects of transvascular exchange and vascular permeability with those of the whole endothelial surface layers. We conclude that many of the barrier properties of the whole surface layer are not representative of the primary fiber matrix forming the molecular filter determining transvascular exchange. The differences between the properties of the whole layer and the inner glycocalyx structures likely reflect dynamic aspects of the endothelial surface layer including tracer binding to specific components, synthesis and degradation of key components, activation of signaling pathways in the endothelial cells when components of the surface layer are lost or degraded, and the spatial distribution of adhesion proteins in microdomains of the endothelial cell membrane.

EICOSAPENTAENOIC ACID ENHANCES HEATSTROKE-IMPAIRED INTESTINAL EPITHELIAL BARRIER FUNCTION IN RATS.

PubMed

Xiao, Guizhen; Yuan, Fangfang; Geng, Yan; Qiu, Xiaowen; Liu, Zhifeng; Lu, Jiefu; Tang, Liqun; Zhang, Yali; Su, Lei

2015-10-01

Dysfunction of the intestinal barrier plays an important role in the pathological process of heatstroke. Omega-3 (or n-3) polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), help protect the intestinal mucosal barrier. This study assessed if pretreating rats with EPA or DHA could alleviate heat stress-induced damage to the intestinal barrier caused by experimental heatstroke. Male Wistar rats were pregavaged with either EPA, DHA, corn oil, or normal saline (all 1 g/kg) for 21 days before the heatstroke experiment (control rats were not exposed to heat). Experimental rats were exposed to an ambient temperature of 37°C and 60% humidity to induce heatstroke, and then they were allowed to recover at room temperature after rapid cooling. Survival time of rats was monitored after heatstroke. Horseradish peroxidase flux from the gut lumen and the level of plasma D-lactate were measured to analyze intestinal permeability at 6 h after heatstroke. Plasma endotoxin levels were determined using a limulus amoebocyte lysate assay. Expressions of the tight junction (TJ) proteins occludin and ZO-1 were analyzed by Western blot and localized by immunofluorescence microscopy. Tight junction protein morphology was observed by transmission electron microscopy. Fatty acids of ileal mucosa were analyzed using gas chromatography-mass selective detector. Eicosapentaenoic acid significantly increased survival time after heatstroke. Eicosapentaenoic acid significantly decreased intestinal permeability and plasma endotoxin levels. Eicosapentaenoic acid effectively attenuated the heatstroke-induced disruption of the intestinal structure and improved the histology score, whereas DHA was less effective, and corn oil was ineffective. Pretreatment with EPA also increased expression of occludin and ZO-1 to effectively prevent TJ disruption. Eicosapentaenoic acid pretreatment enriched itself in the membrane of intestinal cells. Our results indicate that EPA pretreatment is more effective than DHA pretreatment in attenuating heat-induced intestinal dysfunction and preventing TJ damage. Enhanced expression of TJ proteins that support the epithelial barrier integrity may be important for maintaining a functional intestinal barrier during heatstroke.

The subcellular distribution of aquaporin 5 in the cochlea reveals a water shunt at the perilymph-endolymph barrier.

PubMed

Hirt, B; Penkova, Z H; Eckhard, A; Liu, W; Rask-Andersen, H; Müller, M; Löwenheim, H

2010-07-28

Aquaporins are membrane water channel proteins that have also been identified in the cochlea. Auditory function critically depends on the homeostasis of the cochlear fluids perilymph and endolymph. In particular, the ion and water regulation of the endolymph is essential for sensory transduction. Within the cochlear duct the lateral wall epithelium has been proposed to secrete endolymph by an aquaporin-mediated flow of water across its epithelial tight junction barrier. This study identifies interspecies differences in the cellular distribution of aquaporin 5 (AQP5) in the cochlear lateral wall of mice, rats, gerbils and guinea pigs. In addition the cellular expression pattern of AQP5 is described in the human cochlea. Developmental changes in rats demonstrate longitudinal and radial gradients along the cochlear duct. During early postnatal development a pancochlear expression is detected. However a regression to the apical quadrant and limitation to outer sulcus cells (OSCs) is observed in the adult. This developmental loss of AQP5 expression in the basal cochlear segments coincides with a morphological loss of contact between OSCs and the endolymph. At the subcellular level, AQP5 exhibits polarized expression in the apical plasma membrane of the OSCs. Complementary, the basolateral membrane in the root processes of the OSCs exhibits AQP4 expression. This differential localization of AQP5 and AQP4 in the apical and basolateral membranes of the same epithelial cell type suggests a direct aquaporin-mediated transcellular water shunt between the perilymph and endolymph in the OSCs of the cochlear lateral wall. In the human cochlea these findings may have pathophysiological implications attributed to a dysfunctional water regulation by AQP5 such as endolymphatic hydrops (i.e. in Meniere's disease) or sensorineural hearing loss (i.e. in Sjögren's syndrome). Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Infrared and Raman spectroscopic features of plant cuticles: a review

PubMed Central

Heredia-Guerrero, José A.; Benítez, José J.; Domínguez, Eva; Bayer, Ilker S.; Cingolani, Roberto; Athanassiou, Athanassia; Heredia, Antonio

2014-01-01

The cuticle is one of the most important plant barriers. It is an external and continuous lipid membrane that covers the surface of epidermal cells and whose main function is to prevent the massive loss of water. The spectroscopic characterization of the plant cuticle and its components (cutin, cutan, waxes, polysaccharides and phenolics) by infrared and Raman spectroscopies has provided significant advances in the knowledge of the functional groups present in the cuticular matrix and on their structural role, interaction and macromolecular arrangement. Additionally, these spectroscopies have been used in the study of cuticle interaction with exogenous molecules, degradation, distribution of components within the cuticle matrix, changes during growth and development and characterization of fossil plants. PMID:25009549

In vitro evaluation of the human gingival fibroblast/gingival mesenchymal stem cell dynamics through perforated guided tissue membranes: cell migration, proliferation and membrane stiffness assay.

PubMed

Gamal, A Y; Al-Berry, N N; Hassan, A A; Rashed, L A; Iacono, V J

2017-06-01

Migration of gingival fibroblasts/gingival mesenchymal stem cells through macro-perforated barrier membranes may allow them to participate positively in periodontal regeneration. The optimal guided tissue membrane perforation diameter that could favor maximum cell migration into the defect area and at the same time act as an occlusive barrier for gingival epithelium and its associated gingival extracellular matrix component is not yet identified. Cultured human gingival fibroblasts/gingival mesenchymal stem cells were placed in the upper chambers of 12-well collagen-coated polytetrafluoroethylene transwells, which were manually perforated with 0.2, 0.4 and 0.7 mm sized pores. The lower chambers of the transwells received blood clot as an attraction medium. The number of cells that have migrated to the lower chambers was calculated. Proliferation of these cells was evaluated using MTT assay. Scanning electron microscopy images were obtained for the lower surfaces of the transwell membranes. Perforated bovine collagen membranes (Tutopatch ® ) were subjected to mechanical testing to determine the tensile strength and modulus of elasticity. Group 3 (0.7 mm) showed significantly higher values for cell migration and proliferation. All groups showed a small degree of extracellular matrix migration through membrane perforations. Scanning electron microscopy evaluation revealed variable numbers of cells in fibrin matrices located mainly around the pore edges. There were non-significant differences between groups regarding mechanical properties. The present study demonstrated that macro-membrane perforations of 0.2, 0.4 and 0.7 mm are suitable pore diameters that could maintain membrane stiffness and allow for cellular migration. However, these membrane perforation diameters did not allow for total gingival connective tissue isolation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Influence of Cholesterol on the Oxygen Permeability of Membranes: Insight from Atomistic Simulations.

PubMed

Dotson, Rachel J; Smith, Casey R; Bueche, Kristina; Angles, Gary; Pias, Sally C

2017-06-06

Cholesterol is widely known to alter the physical properties and permeability of membranes. Several prior works have implicated cell membrane cholesterol as a barrier to tissue oxygenation, yet a good deal remains to be explained with regard to the mechanism and magnitude of the effect. We use molecular dynamics simulations to provide atomic-resolution insight into the influence of cholesterol on oxygen diffusion across and within the membrane. Our simulations show strong overall agreement with published experimental data, reproducing the shapes of experimental oximetry curves with high accuracy. We calculate the upper-limit transmembrane oxygen permeability of a 1-palmitoyl,2-oleoylphosphatidylcholine phospholipid bilayer to be 52 ± 2 cm/s, close to the permeability of a water layer of the same thickness. With addition of cholesterol, the permeability decreases somewhat, reaching 40 ± 2 cm/s at the near-saturating level of 62.5 mol % cholesterol and 10 ± 2 cm/s in a 100% cholesterol mimic of the experimentally observed noncrystalline cholesterol bilayer domain. These reductions in permeability can only be biologically consequential in contexts where the diffusional path of oxygen is not water dominated. In our simulations, cholesterol reduces the overall solubility of oxygen within the membrane but enhances the oxygen transport parameter (solubility-diffusion product) near the membrane center. Given relatively low barriers to passing from membrane to membrane, our findings support hydrophobic channeling within membranes as a means of cellular and tissue-level oxygen transport. In such a membrane-dominated diffusional scheme, the influence of cholesterol on oxygen permeability is large enough to warrant further attention. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effects of Polymer Structure and Relaxations on Ionic Conductivity in Anion Exchange Membranes with Quaternary Ammonium Functional Groups

NASA Astrophysics Data System (ADS)

Maes, Ashley M.

Anion exchange membranes (AEMs) are of considerable interest to developers and researchers of electrochemical conversion and storage devices such as anion exchange membrane fuel cells (AAEMFCs), alkaline polymer electrolyte electrolysers, redox flow batteries and bioelectrochemical devices. AEMs are generally in competition with more established proton exchange membranes (PEMs), but offer the potential for reduction of materials costs and greater fuel flexibility across these applications. This work includes an introduction to AEMs in the context of fuel cell technologies and some key techniques for AEM characterization. There are many synthetic strategies to incorporate cationic functional groups, which promote anion transport, into a polymer matrix. Two membrane chemistries are investigated in the following chapters. The first is based on a simple synthesis procedure that produced a membrane consisting of random, crosslinked polypropylene- ran-polyethyleneimine with quaternary ammonium functional groups. This membrane had moderate chloride ionic conductivity of 0.03 S cm -1 at 95 °C and high water uptake with minimal dimensional swelling. However, the lack of control of crosslink location and density during synthesis produced a material with a very random nature, making it a poor candidate for more fundamental transport studies. The second membrane chemistry is a block copolymer with a hydrophobic and hydrophilic block. The hydrophobic block was selected to provide favorable mechanical and barrier characteristics while a hydrophilic block was selected to provide water uptake and anion conducting functionalities. Poly(vinyl benzyl trimethyl ammonium bromide)-b-poly(methylbutylene) ([PVBTMA][Br]- b-PMB) was synthesized by partners at the University of Massachusetts-Amherst with varied degrees of functionalization (DF) along the hydrophilic block, resulting in ion exchange capacities ranging from 0.77 to 2.20 mmol g -1. Water uptake, in-plane ionic conductivity and membrane morphology were measured across a series of membranes with the original bromide (Br -) counter-ion. These bulk materials characterization experiments demonstrated that this polymer structure produces well-ordered lamellar morphology with moderate water uptake and competitive ionic conductivity (ca. 40 mS cm-1 at 90 °C and 95% relative humidity). These characteristics make it an appropriate candidate for the following more fundamental investigations of ionic conductivity mechanisms. Broadband electrical spectroscopy (BES) was conducted on one [PVBTMA][Br]- b-PMB sample in the Br- form and analyzed in conjunction with thermal stability and relaxation experiments in Chapter 4. We were able to propose two separate ionic conductivity mechanisms and relate each to physical attributes of the polymer structure. A significant thermal transition was observed at Tdelta , which resulted in a dramatic drop in conductivity. In a continued effort to characterize the ionic conductivity of these block-copolymer membranes, another BES study was conducted on three samples with varying DFs. Samples were converted to hydroxide (OH- ) form so we could contrast the Br- conductivity mechanisms to those in a more relevant counter-ion form. After analysis of the electric response of the material, combined with the thermal analysis by TGA, MDSC and DMA, conductivity mechanisms were described. As in the Br- study, conductivity involves two distinct conduction pathways, sigmaEP and sigmaIP,1. Importantly, we again observed a drop in conductivity at Tdelta in each of these samples, with Tdelta decreasing as the density of functional groups along the hydrophilic block increased. It is undesirable for this transition to occur during operation in a fuel cell or other electrochemical device, so future work to investigate strategies for inhibition are recommended.

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development.

PubMed

Saathoff, Manuela; Blum, Barbara; Quast, Thomas; Kirfel, Gregor; Herzog, Volker

2004-10-01

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier. Copyright 2004 Elsevier Inc.

Impaired Intestinal Mucosal Barrier upon Ischemia-Reperfusion: “Patching Holes in the Shield with a Simple Surgical Method”

PubMed Central

Rosero, Olivér; Ónody, Péter; Molnár, Dávid; Lotz, Gábor; Turóczi, Zsolt; Fülöp, András; Garbaisz, Dávid; Harsányi, László; Szijártó, Attila

2014-01-01

Mesenteric ischemia-reperfusion (IR) is associated with impairment of the gut barrier function and the initiation of a proinflammatory cascade with life-threatening results. Therefore methods directed to ameliorate IR injury are of great importance. We aimed at describing the effects of postconditioning (PC) on the alterations of the intestinal mucosal function and the inflammatory response upon mesenteric IR. Methods. Male Wistar rats were gavaged with green fluorescent protein-expressing E. coli suspensions. Animals were randomized into three groups (n = 15), sham-operated, IR-, and PC-groups, and underwent 60 minutes of superior mesenteric artery occlusion, followed by 6 hours of reperfusion. Postconditioning was performed at the onset of reperfusion. Blood and tissue samples were taken at the end of reperfusion, for histological, bacteriological, and plasma examinations. Results. The PC-group presented a more favorable claudin-2, claudin-3, claudin-4, and zonula occludens-1 membrane expression profile, and significantly lower rates of bacterial translocation to distant organs and plasma D-lactate levels compared to the IR-group. Histopathological lesions, plasma I-FABP, IL-6, and TNF-α levels were significantly lower in the PC-group compared to the IR-group. Conclusion. The use of postconditioning improved the integrity of the intestinal mucosal barrier upon mesenteric IR, and thus reduced the incidence of bacterial translocation and development of a systemic inflammatory response. PMID:24955347

Impaired intestinal mucosal barrier upon ischemia-reperfusion: "patching holes in the shield with a simple surgical method".

PubMed

Rosero, Olivér; Ónody, Péter; Kovács, Tibor; Molnár, Dávid; Lotz, Gábor; Tóth, Szilárd; Turóczi, Zsolt; Fülöp, András; Garbaisz, Dávid; Harsányi, László; Szijártó, Attila

2014-01-01

Mesenteric ischemia-reperfusion (IR) is associated with impairment of the gut barrier function and the initiation of a proinflammatory cascade with life-threatening results. Therefore methods directed to ameliorate IR injury are of great importance. We aimed at describing the effects of postconditioning (PC) on the alterations of the intestinal mucosal function and the inflammatory response upon mesenteric IR. Male Wistar rats were gavaged with green fluorescent protein-expressing E. coli suspensions. Animals were randomized into three groups (n = 15), sham-operated, IR-, and PC-groups, and underwent 60 minutes of superior mesenteric artery occlusion, followed by 6 hours of reperfusion. Postconditioning was performed at the onset of reperfusion. Blood and tissue samples were taken at the end of reperfusion, for histological, bacteriological, and plasma examinations. The PC-group presented a more favorable claudin-2, claudin-3, claudin-4, and zonula occludens-1 membrane expression profile, and significantly lower rates of bacterial translocation to distant organs and plasma D-lactate levels compared to the IR-group. Histopathological lesions, plasma I-FABP, IL-6, and TNF- α levels were significantly lower in the PC-group compared to the IR-group. The use of postconditioning improved the integrity of the intestinal mucosal barrier upon mesenteric IR, and thus reduced the incidence of bacterial translocation and development of a systemic inflammatory response.

Overcoming barriers to membrane protein structure determination.

PubMed

Bill, Roslyn M; Henderson, Peter J F; Iwata, So; Kunji, Edmund R S; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G; Vogel, Horst

2011-04-01

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Pericytes of the neurovascular unit: Key functions and signaling pathways

PubMed Central

Sweeney, Melanie D.; Ayyadurai, Shiva; Zlokovic, Berislav V.

2017-01-01

Pericytes are vascular mural cells embedded in the basement membrane of blood microvessels. They extend their processes along capillaries, pre-capillary arterioles, and post-capillary venules. The central nervous system (CNS) pericytes are uniquely positioned within the neurovascular unit between endothelial cells, astrocytes, and neurons. They integrate, coordinate, and process signals from their neighboring cells to generate diverse functional responses that are critical for CNS functions in health and disease including regulation of the blood-brain barrier permeability, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, neuroinflammation, and stem cell activity. Here, we examine the key signaling pathways between pericytes and their neighboring endothelial cells, astrocytes, and neurons that control neurovascular functions. We also review the role of pericytes in different CNS disorders including rare monogenic diseases and complex neurological disorders such as Alzheimer's disease and brain tumors. Finally, we discuss directions for future studies. PMID:27227366

The Rheological Properties of Lipid Monolayers Modulate the Incorporation of l-Ascorbic Acid Alkyl Esters.

PubMed

Díaz, Yenisleidy de Las Mercedes Zulueta; Mottola, Milagro; Vico, Raquel V; Wilke, Natalia; Fanani, María Laura

2016-01-19

In this work, we tested the hypothesis that the incorporation of amphiphilic drugs into lipid membranes may be regulated by their rheological properties. For this purpose, two members of the l-ascorbic acid alkyl esters family (ASCn) were selected, ASC16 and ASC14, which have different rheological properties when organized at the air/water interface. They are lipophilic forms of vitamin C used in topical pharmacological preparations. The effect of the phase state of the host lipid membranes on ASCn incorporation was explored using Langmuir monolayers. Films of pure lipids with known phase states have been selected, showing liquid-expanded, liquid-condensed, and solid phases as well as pure cholesterol films in liquid-ordered state. We also tested ternary and quaternary mixed films that mimic the properties of cholesterol containing membranes and of the stratum corneum. The compressibility and shear properties of those monolayers were assessed in order to define its phase character. We found that the length of the acyl chain of the ASCn compounds induces differential changes in the rheological properties of the host membrane and subtly regulates the kinetics and extent of the penetration process. The capacity for ASCn uptake was found to depend on the phase state of the host film. The increase in surface pressure resultant after amphiphile incorporation appears to be a function of the capacity of the host membrane to incorporate such amphiphile as well as the rheological response of the film. Hence, monolayers that show a solid phase state responded with a larger surface pressure increase to the incorporation of a comparable amount of amphiphile than liquid-expanded ones. The cholesterol-containing films, including the mixture that mimics stratum corneum, allowed a very scarce ASCn uptake independently of the membrane diffusional properties. This suggests an important contribution of Cho on the maintenance of the barrier function of stratum corneum.

Ni2+ toxicity in rice: effect on membrane functionality and plant water content.

PubMed

Llamas, Andreu; Ullrich, Cornelia I; Sanz, Amparo

2008-10-01

The heavy metal nickel is an essential mineral trace nutrient found at low concentrations in most natural soils. However, it may reach toxic levels in certain areas and affect a number of biochemical and physiological processes in plants. Wilting and leaf necrosis have been described as typical visible symptoms of Ni(2+) toxicity. The plasma membrane (PM) of root cells constitutes the first barrier for the entry of heavy metals but also a target of their toxic action. This work studies the relationship between disturbances of membrane functionality and the development of the typical symptoms of Ni(2+) toxicity. Rice plants (Oryza sativa L. cv. Bahia) grown in nutrient medium containing 0.5mM Ni(2+) showed a significant decrease in water content as a consequence of the stress. Addition of Ni(2+) to the solution bathing the roots induced a concentration-dependent PM depolarization but the activity of the PM-H(+)-ATPase was not inhibited by the presence of Ni(2+) and the initial resting potential recovered in less than 1h. In the short term (hours), membrane permeability of root cells was not significantly affected by Ni(2+) treatments. However, in the long term (days) a drastic loss of K(+) was measured in roots and shoots, which should be responsible for the changes in the water content measured, since stomatal conductance and the transpiration rate remained unaffected by Ni(2+) treatment. The effects induced by Ni(2+) were not permanent and could be reverted, at least in part, by transferring the plants to a medium without Ni(2+).

Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways.

PubMed

Fernández-Calvet, Ariadna; Rodríguez-Arce, Irene; Almagro, Goizeder; Moleres, Javier; Euba, Begoña; Caballero, Lucía; Martí, Sara; Ramos-Vivas, José; Bartholomew, Toby Leigh; Morales, Xabier; Ortíz-de-Solórzano, Carlos; Yuste, José Enrique; Bengoechea, José Antonio; Conde-Álvarez, Raquel; Garmendia, Junkal

2018-05-02

Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.

Controlled transport of latex beads through vertically aligned carbon nanofiber membranes

NASA Astrophysics Data System (ADS)

Zhang, L.; Melechko, A. V.; Merkulov, V. I.; Guillorn, M. A.; Simpson, M. L.; Lowndes, D. H.; Doktycz, M. J.

2002-07-01

Stripes of vertically aligned carbon nanofibers (VACNFs) have been used to form membranes for size selectively controlling the transport of latex beads. Fluidic structures were created in poly(dimethylsiloxane) (PDMS) and interfaced to the VACNF structures for characterization of the membrane pore size. Solutions of fluorescently labeled latex beads were introduced into the PDMS channels and characterized by fluorescence and scanning electron microscopy. Results show that the beads size selectively pass through the nanofiber barriers and the size restriction limit correlates with the interfiber spacing. The results suggest that altering VACNF array density can alter fractionation properties of the membrane. Such membranes may be useful for molecular sorting and for mimicking the properties of natural membranes.

Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork

NASA Astrophysics Data System (ADS)

Sadegh, Sanaz; Higgins, Jenny L.; Mannion, Patrick C.; Tamkun, Michael M.; Krapf, Diego

2017-01-01

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.

Gel-based and gel-free search for plasma membrane proteins in chickpea (Cicer arietinum L.) augments the comprehensive data sets of membrane protein repertoire.

PubMed

Barua, Pragya; Subba, Pratigya; Lande, Nilesh Vikram; Mangalaparthi, Kiran K; Prasad, T S Keshava; Chakraborty, Subhra; Chakraborty, Niranjan

2016-06-30

Plasma membrane (PM) encompasses total cellular contents, serving as semi-porous barrier to cell exterior. This living barrier regulates all cellular exchanges in a spatio-temporal fashion. Most of the essential tasks of PMs including molecular transport, cell-cell interaction and signal transduction are carried out by their proteinaceous components, which make the PM protein repertoire to be diverse and dynamic. Here, we report the systematic analysis of PM proteome of a food legume, chickpea and develop a PM proteome reference map. Proteins were extracted from highly enriched PM fraction of four-week-old seedlings using aqueous two-phase partitioning. To address a population of PM proteins that is as comprehensive as possible, both gel-based and gel-free approaches were employed, which led to the identification of a set of 2732 non-redundant proteins. These included both integral proteins having bilayer spanning domains as well as peripheral proteins associated with PMs through posttranslational modifications or protein-protein interactions. Further, the proteins were subjected to various in-silico analyses and functionally classified based on their gene ontology. Finally an inventory of the complete set of PM proteins, identified in several monocot and dicot species, was created for comparative study with the generated PM protein dataset of chickpea. Chickpea, a rich source of dietary proteins, is the second most cultivated legume, which is grown over 10 million hectares of land worldwide. The annual global production of chickpea hovers around 8.5 million metric tons. Recent chickpea genome sequencing effort has provided a broad genetic basis for highlighting the important traits that may fortify other crop legumes. Improvement in chickpea varieties can further strengthen the world food security, which includes food availability, access and utilization. It is known that the phenotypic trait of a cultivar is the manifestation of the orchestrated functions of its proteins. Study of the PM proteome offers insights into the mechanism of communication between the cell and its environment by identification of receptors, signalling proteins and membrane transporters. Knowledge of the PM protein repertoire of a relatively dehydration tolerant chickpea variety, JG-62, can contribute in development of strategies for metabolic reprograming of crop species and breeding applications. Copyright © 2016 Elsevier B.V. All rights reserved.

ROCK-1 mediates diabetes-induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy.

PubMed

Rothschild, Pierre-Raphaël; Salah, Sawsen; Berdugo, Marianne; Gélizé, Emmanuelle; Delaunay, Kimberley; Naud, Marie-Christine; Klein, Christophe; Moulin, Alexandre; Savoldelli, Michèle; Bergin, Ciara; Jeanny, Jean-Claude; Jonet, Laurent; Arsenijevic, Yvan; Behar-Cohen, Francine; Crisanti, Patricia

2017-08-18

In diabetic retinopathy, the exact mechanisms leading to retinal capillary closure and to retinal barriers breakdown remain imperfectly understood. Rho-associated kinase (ROCK), an effector of the small GTPase Rho, involved in cytoskeleton dynamic regulation and cell polarity is activated by hyperglycemia. In one year-old Goto Kakizaki (GK) type 2 diabetic rats retina, ROCK-1 activation was assessed by its cellular distribution and by phosphorylation of its substrates, MYPT1 and MLC. In both GK rat and in human type 2 diabetic retinas, ROCK-1 is activated and associated with non-apoptotic membrane blebbing in retinal vessels and in retinal pigment epithelium (RPE) that respectively form the inner and the outer barriers. Activation of ROCK-1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. In RPE cells, actin cytoskeleton remodeling and membrane blebs in RPE cells contributes to outer barrier breakdown. Intraocular injection of fasudil, significantly reduces both retinal hypoxia and RPE barrier breakdown. Diabetes-induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.

Two barriers for sodium in vascular endothelium?

PubMed Central

Oberleithner, Hans

2012-01-01

Vascular endothelium plays a key role in blood pressure regulation. Recently, it has been shown that a 5% increase of plasma sodium concentration (sodium excess) stiffens endothelial cells by about 25%, leading to cellular dysfunction. Surface measurements demonstrated that the endothelial glycocalyx (eGC), an anionic biopolymer, deteriorates when sodium is elevated. In view of these results, a two-barrier model for sodium exiting the circulation across the endothelium is suggested. The first sodium barrier is the eGC which selectively buffers sodium ions with its negatively charged prote-oglycans.The second sodium barrier is the endothelial plasma membrane which contains sodium channels. Sodium excess, in the presence of aldosterone, leads to eGC break-down and, in parallel, to an up-regulation of plasma membrane sodium channels. The following hypothesis is postulated: Sodium excess increases vascular sodium permeability. Under such con-ditions (e.g. high-sodium diet), day-by-day ingested sodium, instead of being readily buffered by the eGC and then rapidly excreted by the kidneys, is distributed in the whole body before being finally excreted. Gradually, the sodium overload damages the organism. PMID:22471931

Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli.

PubMed

Joshi, Suresh G; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D

2011-03-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.

Nonthermal Dielectric-Barrier Discharge Plasma-Induced Inactivation Involves Oxidative DNA Damage and Membrane Lipid Peroxidation in Escherichia coli▿

PubMed Central

Joshi, Suresh G.; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K.; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D.

2011-01-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria. PMID:21199923

Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

PubMed Central

Ader, Nadine; Brindley, Melinda; Avila, Mislay; Örvell, Claes; Horvat, Branka; Hiltensperger, Georg; Schneider-Schaulies, Jürgen; Vandevelde, Marc; Zurbriggen, Andreas; Plemper, Richard K.

2013-01-01

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering. PMID:23077316

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

PubMed

Plesnar, Elzbieta; Szczelina, Robert; Subczynski, Witold K; Pasenkiewicz-Gierula, Marta

2018-02-01

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (P M ) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the P M across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the P M of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood-brain barrier.

PubMed

Volle, Romain; Archimbaud, Christine; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Mirand, Audrey; Pereira, Bruno; Henquell, Cécile; Peigue-Lafeuille, Hélène; Bailly, Jean-Luc

2015-07-01

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.

Drug Delivery Through the Skin: Molecular Simulations of Barrier Lipids to Design more Effective Noninvasive Dermal and Transdermal Delivery Systems for Small Molecules Biologics and Cosmetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Torin Huzil; S Sivaloganathan; M Kohandel

The delivery of drugs through the skin provides a convenient route of administration that is often preferable to injection because it is noninvasive and can typically be self-administered. These two factors alone result in a significant reduction of medical complications and improvement in patient compliance. Unfortunately, a significant obstacle to dermal and transdermal drug delivery alike is the resilient barrier that the epidermal layers of the skin, primarily the stratum corneum, presents for the diffusion of exogenous chemical agents. Further advancement of transdermal drug delivery requires the development of novel delivery systems that are suitable for modern, macromolecular protein andmore » nucleotide therapeutic agents. Significant effort has already been devoted to obtain a functional understanding of the physical barrier properties imparted by the epidermis, specifically the membrane structures of the stratum corneum. However, structural observations of membrane systems are often hindered by low resolutions, making it difficult to resolve the molecular mechanisms related to interactions between lipids found within the stratum corneum. Several models describing the molecular diffusion of drug molecules through the stratum corneum have now been postulated, where chemical permeation enhancers are thought to disrupt the underlying lipid structure, resulting in enhanced permeability. Recent investigations using biphasic vesicles also suggested a possibility for novel mechanisms involving the formation of complex polymorphic lipid phases. In this review, we discuss the advantages and limitations of permeation-enhancing strategies and how computational simulations, at the atomic scale, coupled with physical observations can provide insight into the mechanisms of diffusion through the stratum corneum.« less

Mechanisms of mineral membrane fouling growth modulated by pulsed modes of current during electrodialysis: evidences of water splitting implications in the appearance of the amorphous phases of magnesium hydroxide and calcium carbonate.

PubMed

Cifuentes-Araya, Nicolás; Astudillo-Castro, Carolina; Bazinet, Laurent

2014-07-15

Experiments revealed the fouling nature evolutions along different electrodialysis (ED) trials, and how it disappears when current pulsation acts repetitively on the interfaces of ion-exchange membranes (IEMs). Fouling was totally controlled on the diluate side of cation-exchange membrane (CEM) by the repetitive pulsation frequency of the higher on-duty ratios applied. They created steady water splitting proton-barriers that neutralized OH(-) leakage through the membrane, decreasing the interfacial pH, and fouling of the concentrate side. The anion-exchange membrane (AEM) on the diluate side was similarly protected, but it was fouled once water splitting OH(-) generation became either intense enough or excessively weak. Interestingly, amorphous magnesium hydroxide (AMH) stemmed on the CEM-diluate side from brucite under intense water splitting OH(-) generation, and/or strong OH(-) leakage electromigration through the membrane. Water dissociation and overlimiting current regimes triggered drastic water molecule removal from crystal lattices through an accelerated cascade water splitting reaction. Also, amorphous calcium carbonate (ACC) appeared on CEM under intense water splitting reaction, and disappeared once intense OH(-) leakage was allowed by the water splitting proton-barrier dissipation. Our findings have implications for membrane fouling control, as well as for the understanding of the growth behavior of CaCO3 and Mg(OH)2 species on electromembrane interfaces. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of the effects of hyaluronic acid-carboxymethyl cellulose barrier on ovarian tumor progression

PubMed Central

2014-01-01

Background Hyaluronic acid is a prognostic factor in ovarian cancers. It is also a component of Hyaluronic Acid-Carboxymethyl Cellulose (HA-CMC) barrier, an anti-adhesion membrane widely used during abdominal surgeries in particular for ovarian carcinosis. 70% of patients who undergo ovarian surgery will relapse due to the persistence of cancer cells. This study’s objective was to determine the oncological risk from use of this material, in the presence of residual disease, despite the benefit gained by it decreasing post-surgical adhesions in order to provide an unambiguous assessment of its appropriateness for use in ovarian surgical management. Methods We assessed the effects of HA-CMC barrier on the in vitro proliferation of human ovarian tumor cell lines (OVCAR-3, IGROV-1 and SKOV-3). We next evaluated, in vivo in nude mice, the capacity of this biomaterial to regulate the tumor progression of subcutaneous and intraperitoneal models of ovarian tumor xenografts. Results We showed that HA-CMC barrier does not increase in vitro proliferation of ovarian cancer cell lines compared to control. In vivo, HA-CMC barrier presence with subcutaneous xenografts induced neither an increase in tumor volume nor cell proliferation (Ki67 and mitotic index). With the exception of an increased murine carcinosis score in peritoneum, the presence of HA-CMC barrier with intraperitoneal xenografts modified neither macro nor microscopic tumor growth. Finally, protein analysis of survival (Akt), proliferation (ERK) and adhesion (FAK) pathways highlighted no activation on the xenografts imputable to HA-CMC barrier. Conclusions For the most part, our results support the lack of tumor progression activation due to HA-CMC barrier. We conclude that the benefits gained from using HA-CMC barrier membrane during ovarian cancer surgeries seem to outweigh the potential oncological risks. PMID:24739440

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

PubMed

Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette; Stensballe, Allan; Moos, Torben

2017-03-01

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)] 2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789. © 2016 International Society for Neurochemistry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lv, Chao; Aitchison, Erick W.; Wu, Dongsheng

Hydrogen sulfide (H 2S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H 2S and its structural analogue, water (H 2O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. Here, as revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is,more » the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H 2S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. Lastly, this study not only explains why H 2S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes.« less

Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model skin lipid membranes: permeability and biophysics.

PubMed

Školová, Barbora; Kováčik, Andrej; Tesař, Ondřej; Opálka, Lukáš; Vávrová, Kateřina

2017-05-01

Ceramides based on phytosphingosine, sphingosine and dihydrosphingosine are essential constituents of the skin lipid barrier that protects the body from excessive water loss. The roles of the individual ceramide subclasses in regulating skin permeability and the reasons for C4-hydroxylation of these sphingolipids are not completely understood. We investigated the chain length-dependent effects of dihydroceramides, sphingosine ceramides (with C4-unsaturation) and phytoceramides (with C4-hydroxyl) on the permeability, lipid organization and thermotropic behavior of model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesteryl sulfate. Phytoceramides with very long C24 acyl chains increased the permeability of the model lipid membranes compared to dihydroceramides or sphingosine ceramides with the same chain lengths. Either unsaturation or C4-hydroxylation of dihydroceramides induced chain length-dependent increases in membrane permeability. Infrared spectroscopy showed that C4-hydroxylation of the sphingoid base decreased the relative ratio of orthorhombic chain packing in the membrane and lowered the miscibility of C24 phytoceramide with lignoceric acid. The phase separation in phytoceramide membranes was confirmed by X-ray diffraction. In contrast, phytoceramides formed strong hydrogen bonds and highly thermostable domains. Thus, the large heterogeneity in ceramide structures and in their aggregation mechanisms may confer resistance towards the heterogeneous external stressors that are constantly faced by the skin barrier. Copyright © 2017 Elsevier B.V. All rights reserved.

A tethering complex drives the terminal stage of SNARE-dependent membrane fusion

NASA Astrophysics Data System (ADS)

D'Agostino, Massimo; Risselada, Herre Jelger; Lürick, Anna; Ungermann, Christian; Mayer, Andreas

2017-11-01

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore. This last step is highly energy-demanding. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.

Acyl-Lipid Metabolism

PubMed Central

Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

2013-01-01

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:23505340

Acyl-Lipid Metabolism

PubMed Central

Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

2010-01-01

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:22303259

Low Dielectric Permittivity of Water at the Membrane Interface: Effect on the Energy Coupling Mechanism in Biological Membranes

PubMed Central

Cherepanov, Dmitry A.; Feniouk, Boris A.; Junge, Wolfgang; Mulkidjanian, Armen Y.

2003-01-01

Protonmotive force (the transmembrane difference in electrochemical potential of protons, \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\Delta}{\\tilde {{\\mu}}}_{{\\mathrm{H}}^{+}}\\end{equation*}\\end{document}) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\Delta}{\\tilde {{\\mu}}}_{{\\mathrm{H}}^{+}}\\end{equation*}\\end{document} relates to the difference in the proton activity between two bulk water phases (ΔpHB) or between two membrane surfaces (ΔpHS). To scrutinize whether ΔpHS can deviate from ΔpHB, we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, Phys. Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5–1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2–0.3 eV) than for monovalent cations (0.1–0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton “pumps” at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pHS of ∼6.0; the value of pHS was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria. PMID:12885673

Modulation of Intestinal Paracellular Transport by Bacterial Pathogens.

PubMed

Roxas, Jennifer Lising; Viswanathan, V K

2018-03-25

The passive and regulated movement of ions, solutes, and water via spaces between cells of the epithelial monolayer plays a critical role in the normal intestinal functioning. This paracellular pathway displays a high level of structural and functional specialization, with the membrane-spanning complexes of the tight junctions, adherens junctions, and desmosomes ensuring its integrity. Tight junction proteins, like occludin, tricellulin, and the claudin family isoforms, play prominent roles as barriers to unrestricted paracellular transport. The past decade has witnessed major advances in our understanding of the architecture and function of epithelial tight junctions. While it has been long appreciated that microbes, notably bacterial and viral pathogens, target and disrupt junctional complexes and alter paracellular permeability, the precise mechanisms remain to be defined. Notably, renewed efforts will be required to interpret the available data on pathogen-mediated barrier disruption in the context of the most recent findings on tight junction structure and function. While much of the focus has been on pathogen-induced dysregulation of junctional complexes, commensal microbiota and their products may influence paracellular permeability and contribute to the normal physiology of the gut. Finally, microbes and their products have become important tools in exploring host systems, including the junctional properties of epithelial cells. © 2018 American Physiological Society. Compr Physiol 8:823-842, 2018. Copyright © 2018 American Physiological Society. All rights reserved.

Role of computer-assisted analysis of the corneal endothelium in vitreoretinal surgery with intraocular silicone oil injection: a technical report.

PubMed

Fruschelli, M; Esposti, P; Caporossi, A

1998-01-01

The innermost lining of the cornea consists of a single layer of cells called the endothelium. Despite its name, the endothelium of the cornea differs considerably from the vascular endothelium, both functionally and morphologically. The corneal endothelium plays a fundamental role in maintaining the transparency of the corneal membrane, as the result of both its function as a barrier against penetration of the aqueous humor in the parenchyma and its ability to remove water from the stroma (usually referred to as the endothelial "pump" function). Any abnormality in the corneal endothelium causes, first, the impairment of its function as a barrier and pump due to the loss of stromal anti-turgor mechanisms, followed by edema and possible development into keratopathy. The specular microscope is an instrument which makes it possible to see the endothelial "mosaic" in the reflected image of the posterior corneal surface. A large variety of clinical specular microscopes is presently available, both contact and non-contact, which allow, for easy and rapid photography of the corneal endothelium "in vivo". In the present case, we used a non-contact computerized specular microscope to analyze the corneal endothelium in a group of patients affected by retinal detachment who needed to undergo vitreoretinal surgery with immission of silicone oil into the vitreal chamber.

Coordination of Septate Junctions Assembly and Completion of Cytokinesis in Proliferative Epithelial Tissues.

PubMed

Daniel, Emeline; Daudé, Marion; Kolotuev, Irina; Charish, Kristi; Auld, Vanessa; Le Borgne, Roland

2018-05-07

How permeability barrier function is maintained when epithelial cells divide is largely unknown. Here, we have investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. We report that, following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. We propose that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia. Copyright © 2018 Elsevier Ltd. All rights reserved.

Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation.

PubMed

Bae, Hyoung Eun; Gotfryd, Kamil; Thomas, Jennifer; Hussain, Hazrat; Ehsan, Muhammad; Go, Juyeon; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

2015-07-06

Detergents are an absolute requirement for studying the structure of membrane proteins. However, many conventional detergents fail to stabilise denaturation-sensitive membrane proteins, such as eukaryotic proteins and membrane protein complexes. New amphipathic agents with enhanced efficacy in stabilising membrane proteins will be helpful in overcoming the barriers to studying membrane protein structures. We have prepared a number of deoxycholate-based amphiphiles with carbohydrate head groups, designated deoxycholate-based glycosides (DCGs). These DCGs are the hydrophilic variants of previously reported deoxycholate-based N-oxides (DCAOs). Membrane proteins in these agents, particularly the branched diglucoside-bearing amphiphiles DCG-1 and DCG-2, displayed favourable behaviour compared to previously reported parent compounds (DCAOs) and conventional detergents (LDAO and DDM). Given their excellent properties, these agents should have significant potential for membrane protein studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultra-thin solid oxide fuel cells: Materials and devices

NASA Astrophysics Data System (ADS)

Kerman, Kian

Solid oxide fuel cells are electrochemical energy conversion devices utilizing solid electrolytes transporting O2- that typically operate in the 800 -- 1000 °C temperature range due to the large activation barrier for ionic transport. Reducing electrolyte thickness or increasing ionic conductivity can enable lower temperature operation for both stationary and portable applications. This thesis is focused on the fabrication of free standing ultrathin (<100 nm) oxide membranes of prototypical O 2- conducting electrolytes, namely Y2O3-doped ZrO2 and Gd2O3-doped CeO2. Fabrication of such membranes requires an understanding of thin plate mechanics coupled with controllable thin film deposition processes. Integration of free standing membranes into proof-of-concept fuel cell devices necessitates ideal electrode assemblies as well as creative processing schemes to experimentally test devices in a high temperature dual environment chamber. We present a simple elastic model to determine stable buckling configurations for free standing oxide membranes. This guides the experimental methodology for Y 2O3-doped ZrO2 film processing, which enables tunable internal stress in the films. Using these criteria, we fabricate robust Y2O3-doped ZrO2 membranes on Si and composite polymeric substrates by semiconductor and micro-machining processes, respectively. Fuel cell devices integrating these membranes with metallic electrodes are demonstrated to operate in the 300 -- 500 °C range, exhibiting record performance at such temperatures. A model combining physical transport of electronic carriers in an insulating film and electrochemical aspects of transport is developed to determine the limits of performance enhancement expected via electrolyte thickness reduction. Free standing oxide heterostructures, i.e. electrolyte membrane and oxide electrodes, are demonstrated. Lastly, using Y2O3-doped ZrO2 and Gd2O 3-doped CeO2, novel electrolyte fabrication schemes are explored to develop oxide alloys and nanoscale compositionally graded membranes that are thermomechanically robust and provide added interfacial functionality. The work in this thesis advances experimental state-of-the-art with respect to solid oxide fuel cell operation temperature, provides fundamental boundaries expected for ultrathin electrolytes, develops the ability to integrate highly dissimilar material (such as oxide-polymer) heterostructures, and introduces nanoscale compositionally graded electrolyte membranes that can lead to monolithic materials having multiple functionalities.

Hampering Effect of Cholesterol on the Permeation of Reactive Oxygen Species through Phospholipids Bilayer: Possible Explanation for Plasma Cancer Selectivity

NASA Astrophysics Data System (ADS)

van der Paal, Jonas; Verheyen, Claudia; Neyts, Erik C.; Bogaerts, Annemie

2017-01-01

In recent years, the ability of cold atmospheric pressure plasmas (CAPS) to selectively induce cell death in cancer cells has been widely established. This selectivity has been assigned to the reactive oxygen and nitrogen species (RONS) created in CAPs. To provide new insights in the search for an explanation for the observed selectivity, we calculate the transfer free energy of multiple ROS across membranes containing a varying amount of cholesterol. The cholesterol fraction is investigated as a selectivity parameter because membranes of cancer cells are known to contain lower fractions of cholesterol compared to healthy cells. We find that cholesterol has a significant effect on the permeation of reactive species across a membrane. Indeed, depending on the specific reactive species, an increasing cholesterol fraction can lead to (i) an increase of the transfer free energy barrier height and width, (ii) the formation of a local free energy minimum in the center of the membrane and (iii) the creation of extra free energy barriers due to the bulky sterol rings. In the context of plasma oncology, these observations suggest that the increased ingress of RONS in cancer cells can be explained by the decreased cholesterol fraction of their cell membrane.

Membrane Fusion Induced by Small Molecules and Ions

PubMed Central

Mondal Roy, Sutapa; Sarkar, Munna

2011-01-01

Membrane fusion is a key event in many biological processes. These processes are controlled by various fusogenic agents of which proteins and peptides from the principal group. The fusion process is characterized by three major steps, namely, inter membrane contact, lipid mixing forming the intermediate step, pore opening and finally mixing of inner contents of the cells/vesicles. These steps are governed by energy barriers, which need to be overcome to complete fusion. Structural reorganization of big molecules like proteins/peptides, supplies the required driving force to overcome the energy barrier of the different intermediate steps. Small molecules/ions do not share this advantage. Hence fusion induced by small molecules/ions is expected to be different from that induced by proteins/peptides. Although several reviews exist on membrane fusion, no recent review is devoted solely to small moleculs/ions induced membrane fusion. Here we intend to present, how a variety of small molecules/ions act as independent fusogens. The detailed mechanism of some are well understood but for many it is still an unanswered question. Clearer understanding of how a particular small molecule can control fusion will open up a vista to use these moleucles instead of proteins/peptides to induce fusion both in vivo and in vitro fusion processes. PMID:21660306

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

PubMed

Zhang, Yongli

2017-07-01

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.

The urothelium of a hibernator: the American black bear

PubMed Central

Spector, David A; Deng, Jie; Coleman, Richard; Wade, James B

2015-01-01

The American black bear undergoes a 3–5 month winter hibernation during which time bears do not eat, drink, defecate, or urinate. During hibernation renal function (GFR) is 16–50% of normal but urine is reabsorbed across the urinary bladder (UB) urothelium thus enabling metabolic recycling of all urinary constituents. To elucidate the mechanism(s) whereby urine is reabsorbed, we examined the UBs of five nonhibernating wild bears using light, electron (EM), and confocal immunofluorescent (IF) microscopy–concentrating on two components of the urothelial permeability barrier – the umbrella cell apical membranes and tight junctions (TJ). Bear UB has the same tissue layers (serosa, muscularis, lamina propria, urothelia) and its urothelia has the same cell layers (basal, intermediate, umbrella cells) as other mammalians. By EM, the bear apical membrane demonstrated a typical mammalian scalloped appearance with hinge and plaque regions – the latter containing an asymmetric trilaminar membrane and, on IF, uroplakins Ia, IIIa, and IIIb. The umbrella cell TJs appeared similar to those in other mammals and also contained TJ proteins occludin and claudin - 4, and not claudin –2. Thus, we were unable to demonstrate urothelial apical membrane or TJ differences between active black bears and other mammals. Expression and localization of UT-B, AQP-1 and -3, and Na+, K+-ATPase on bear urothelial membranes was similar to that of other mammals. Similar studies of urothelia of hibernating bears, including evaluation of the apical membrane lipid bilayer and GAGs layer are warranted to elucidate the mechanism(s) whereby hibernating bears reabsorb their daily urine output and thus ensure successful hibernation. PMID:26109187

Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

PubMed

Narayan, Shoba; Devi, R S; Devi, C S Shyamala

2007-11-20

Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.

Peroxisomal membrane permeability and solute transfer.

PubMed

Antonenkov, Vasily D; Hiltunen, J Kalervo

2006-12-01

The review is dedicated to recent progress in the study of peroxisomal membrane permeability to solutes which has been a matter of debate for more than 40 years. Apparently, the mammalian peroxisomal membrane is freely permeable to small solute molecules owing to the presence of pore-forming channels. However, the membrane forms a permeability barrier for 'bulky' solutes including cofactors (NAD/H, NADP/H, CoA, and acetyl/acyl-CoA esters) and ATP. Therefore, peroxisomes need specific protein transporters to transfer these compounds across the membrane. Recent electrophysiological studies have revealed channel-forming activities in the mammalian peroxisomal membrane. The possible involvement of the channels in the transfer of small metabolites and in the formation of peroxisomal shuttle systems is described.

A preliminary study of aquaporin 1 immunolocalization in chronic subdural hematoma membranes.

PubMed

Basaldella, Luca; Perin, Alessandro; Orvieto, Enrico; Marton, Elisabetta; Itskevich, David; Dei Tos, Angelo Paolo; Longatti, Pierluigi

2010-07-01

Aquaporin 1 (AQP1) is a molecular water channel expressed in many anatomical locations, particularly in epithelial barriers specialized in water transport. The aim of this study was to investigate AQP1 expression in chronic subdural hematoma (CSDH) membranes. In this preliminary study, 11 patients with CSDH underwent burr hole craniectomy and drainage. Membrane specimens were stained with a monoclonal antibody targeting AQP1 for immunohistochemical analysis. The endothelial cells of the sinusoid capillaries of the outer membranes exhibited an elevated immunoreactivity to AQP1 antibody compared to the staining intensity of specimens from the inner membrane and normal dura. These findings suggest that the outer membrane might be the source of the increased fluid accumulation responsible for chronic hematoma enlargement.

Implicit membrane treatment of buried charged groups: application to peptide translocation across lipid bilayers.

PubMed

Lazaridis, Themis; Leveritt, John M; PeBenito, Leo

2014-09-01

The energetic cost of burying charged groups in the hydrophobic core of lipid bilayers has been controversial, with simulations giving higher estimates than certain experiments. Implicit membrane approaches are usually deemed too simplistic for this problem. Here we challenge this view. The free energy of transfer of amino acid side chains from water to the membrane center predicted by IMM1 is reasonably close to all-atom free energy calculations. The shape of the free energy profile, however, for the charged side chains needs to be modified to reflect the all-atom simulation findings (IMM1-LF). Membrane thinning is treated by combining simulations at different membrane widths with an estimate of membrane deformation free energy from elasticity theory. This approach is first tested on the voltage sensor and the isolated S4 helix of potassium channels. The voltage sensor is stably inserted in a transmembrane orientation for both the original and the modified model. The transmembrane orientation of the isolated S4 helix is unstable in the original model, but a stable local minimum in IMM1-LF, slightly higher in energy than the interfacial orientation. Peptide translocation is addressed by mapping the effective energy of the peptide as a function of vertical position and tilt angle, which allows identification of minimum energy pathways and transition states. The barriers computed for the S4 helix and other experimentally studied peptides are low enough for an observable rate. Thus, computational results and experimental studies on the membrane burial of peptide charged groups appear to be consistent. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

PubMed

Richards, Mark J; Daniel, Susan

2017-02-07

The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

Self assembly properties of primitive organic compounds

NASA Technical Reports Server (NTRS)

Deamer, D. W.

1991-01-01

A central event in the origin of life was the self-assembly of amphiphilic, lipid-like compounds into closed microenvironments. If a primitive macromolecular replicating system could be encapsulated within a vesicular membrane, the components of the system would share the same microenvironment, and the result would be a step toward true cellular function. The goal of our research has been to determine what amphiphilic molecules might plausibly have been available on the early Earth to participate in the formation of such boundary structures. To this end, we have investigated primitive organic mixtures present in carbonaceous meteorites such as the Murchison meteorite, which contains 1-2 percent of its mass in the form of organic carbon compounds. It is likely that such compounds contributed to the inventory of organic carbon on the prebiotic earth, and were available to participate in chemical evolution leading to the emergence of the first cellular life forms. We found that Murchison components extracted into non-polar solvent systems are surface active, a clear indication of amphiphilic character. One acidic fraction self-assembles into vesicular membranes that provide permeability barriers to polar solutes. Other evidence indicates that the membranes are bimolecular layers similar to those formed by contemporary membrane lipids. We conclude that bilayer membrane formation by primitive amphiphiles on the early Earth is feasible. However, only a minor fraction of acidic amphiphiles assembles into bilayers, and the resulting membranes require narrowly defined conditions of pH and ionic composition to be stable. It seems unlikely, therefore, that meteoritic infall was a direct source of membrane amphiphiles. Instead, the hydrocarbon components and their derivatives more probably would provide an organic stock available for chemical evolution. Our current research is directed at possible reactions which would generate substantial quantities of membranogenic amphiphiles. One possibility is photochemical oxidation of hydrocarbons.

Permeability of membranes to amino acids and modified amino acids: mechanisms involved in translocation

NASA Technical Reports Server (NTRS)

Chakrabarti, A. C.; Deamer, D. W. (Principal Investigator); Miller, S. L. (Principal Investigator)

1994-01-01

The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10(-12)-10(-13) cm s-1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10(2) more permeable than the hydrophilic forms, reflecting their increased partition coefficient values. External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10(-2) cm s-1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.

Electrospun F18 Bioactive Glass/PCL—Poly (ε-caprolactone)—Membrane for Guided Tissue Regeneration

PubMed Central

Hidalgo Pitaluga, Lucas; Trevelin Souza, Marina; Santocildes Romero, Martin Eduardo; Hatton, Paul V.

2018-01-01

Barrier membranes that are used for guided tissue regeneration (GTR) therapy usually lack bioactivity and the capability to promote new bone tissue formation. However, the incorporation of an osteogenic agent into polymeric membranes seems to be the most assertive strategy to enhance their regenerative potential. Here, the manufacturing of composite electrospun membranes made of poly (ε-caprolactone) (PCL) and particles of a novel bioactive glass composition (F18) is described. The membranes were mechanically and biologically tested with tensile strength tests and tissue culture with MG-63 osteoblast-like cell line, respectively. The PCL-F18 composite membranes demonstrated no increased cytotoxicity and an enhanced osteogenic potential when compared to pure PCL membranes. Moreover, the addition of the bioactive phase increased the membrane tensile strength. These preliminary results suggested that these new membranes can be a strong candidate for small bone injuries treatment by GTR technique. PMID:29517988

Prenatal exposure to common environmental factors affects brain lipids and increases risk of developing autism spectrum disorders.

PubMed

Wong, Christine T; Wais, Joshua; Crawford, Dorota A

2015-11-01

The prevalence of autism spectrum disorders (ASDs) has been on the rise over recent years. The presence of diverse subsets of candidate genes in each individual with an ASD and the vast variability of phenotypical differences suggest that the interference of an exogenous environmental component may greatly contribute to the development of ASDs. The lipid mediator prostaglandin E2 (PGE2 ) is released from phospholipids of cell membranes, and is important in brain development and function; PGE2 is involved in differentiation, synaptic plasticity and calcium regulation. The previous review already described extrinsic factors, including deficient dietary supplementation, and exposure to oxidative stress, infections and inflammation that can disrupt signaling of the PGE2 pathway and contribute to ASDs. In this review, the structure and establishment of two key protective barriers for the brain during early development are described: the blood-brain barrier; and the placental barrier. Then, the first comprehensive summary of other environmental factors, such as exposure to chemicals in air pollution, pesticides and consumer products, which can also disturb PGE2 signaling and increase the risk for developing ASDs is provided. Also, how these exogenous agents are capable of crossing the protective barriers of the brain during critical developmental periods when barrier components are still being formed is described. This review underlines the importance of avoiding or limiting exposure to these factors during vulnerable periods in development. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

A viable circulating tumor cell isolation device with high retrieval efficiency using a reversibly deformable membrane barrier

NASA Astrophysics Data System (ADS)

Kim, Yoonji; Bu, Jiyoon; Cho, Young-Ho; Son, Il Tae; Kang, Sung-Bum

2017-02-01

Circulating tumor cells (CTCs) contain prognostic information of the tumor, since they shed from the primary tumor and invade into the bloodstream. Therefore, the viable isolation is necessary for a consequent analysis of CTCs. Here, we present a device for the viable isolation and efficient retrieval of CTCs using slanted slot filters, formed by a reversibly deformable membrane barrier. Conventional filters have difficulties in retrieving captured cells, since they easily clog the slots. Moreover, large stress concentration at the sharp edges of squared slots, causes cell lysis. In contrast, the present device shows over 94% of high retrieval efficiency, since the slots can be opened simply by relieving the pressure. Furthermore, the inflated membrane barrier naturally forms the slanted slots, thus reducing the cell damage. By using cancer cell lines, we verified that the present device successfully isolate targeted cells, even at an extremely low concentrations (~10 cells/0.1 ml). In the clinical study, 85.7% of patients initially showed CTC positive while the numbers generally decreased after the surgery. We have also proved that the number of CTCs were highly correlated with tumour invasiveness. Therefore, the present device has potential for use in cancer diagnosis, surgical validation, and invasiveness analysis.

Atomic Force Microscopy of Biological Membranes

PubMed Central

Frederix, Patrick L.T.M.; Bosshart, Patrick D.; Engel, Andreas

2009-01-01

Abstract Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes. PMID:19167286

How the spontaneous insertion of amphiphilic imidazolium-based cations changes biological membranes: a molecular simulation study.

PubMed

Lim, Geraldine S; Jaenicke, Stephan; Klähn, Marco

2015-11-21

The insertion of 1-octyl-3-methylimidazolium cations (OMIM(+)) from a diluted aqueous ionic liquid (IL) solution into a model of a bacterial cell membrane is investigated. Subsequently, the mutual interactions of cations inside the membrane and their combined effect on membrane properties are derived. The ionic liquid solution and the membrane model are simulated using molecular dynamics in combination with empirical force fields. A high propensity of OMIM(+) for membrane insertion is observed, with a cation concentration at equilibrium inside the membrane 47 times larger than in the solvent. Once inserted, cations exhibit a weak effective attraction inside the membrane at a distance of 1.3 nm. At this free energy minimum, negatively charged phosphates of the phospholipids are sandwiched between two OMIM(+) to form energetically favorable OMIM(+)-phosphate-OMIM(+) types of coordination. The cation-cation association free energy is 5.9 kJ mol(-1), whereas the activation barrier for dissociation is 10.1 kJ mol(-1). Subsequently, OMIM(+) are inserted into the leaflet of the membrane bilayer that represents the extracellular side. The cations are evenly distributed with mutual cation distances according to the found optimum distance of 1.3 nm. Because of the short length of the cation alkyl chains compared to lipid fatty acids, voids are generated in the hydrophobic core of the membrane. These voids disorder the fatty acids, because they enable fatty acids to curl into these empty spaces and also cause a thinning of the membrane by 0.6 nm. Additionally, the membrane density increases at its center. The presence of OMIM(+) in the membrane facilitates the permeation of small molecules such as ammonia through the membrane, which is chosen as a model case for small polar solutes. The permeability coefficient of the membrane with respect to ammonia increases substantially by a factor of seven. This increase is caused by a reduction of the involved free energy barriers, which is effected by the cations through the thinning of the membrane and favorable interactions of the delocalized OMIM(+) charge with ammonia inside the membrane. Overall, the results indicate the antimicrobial effect of amphiphilic imidazolium-based cations that are found in various common ILs. This effect is caused by an alteration of the permeability of the bacterial membrane and other property changes.

Role of Sigma-1 Receptor in Cocaine Abuse and Neurodegenerative Disease.

PubMed

Cai, Yu; Yang, Lu; Niu, Fang; Liao, Ke; Buch, Shilpa

2017-01-01

Sigma-1 receptors (Sig-1R) are recognized as a unique class of non-G protein-coupled intracellular protein. Sig-1R binds to its ligand such as cocaine , resulting in dissociation of Sig-1R from mitochondrion-associated ER membrane (MAM) to the endoplasmic reticulum (ER), plasma membrane, and nuclear membrane, regulating function of various proteins. Sig-1R has diverse roles in both physiological as well as in pathogenic processes. The disruption of Sig-1R pathways has been implicated as causative mechanism(s) in the development of both neurodegenerative disorders such as Alzheimer disease (AD ), Parkinson disease (PD ), amyotrophic lateral sclerosis (ALS ) and Huntington Disease (HD ) . Additionally, the interaction of cocaine and Sig-1R has more recently been implicated in potentiating the pathogenesis of HIV-associated neurocognitive disorders (HAND) through impairment of blood-brain barrier (BBB), microglial activation and astrogliosis. On the other hand, restoration of Sig-1R homeostasis has been shown to exert neuroprotective effects. In this review, we provide an overview of how Sig-1R plays a role in the pathogenesis of neurodegenerative disorders and cocaine and implications for future development of therapeutic strategies.

Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2)more » after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP2A/MMP-2 induced PCB153-induced dysfunction of occludin. • Disrupted lipid rafts modulated PCB153-induced increase of permeability. • Lipid rafts act as a signaling platform for PCB153-induced dysfunction of occludin.« less

Extracorporeal membrane oxygenation and cytokine adsorption

PubMed Central

Träger, Karl

2018-01-01

Extracorporeal membrane oxygenation (ECMO) is an increasingly used technology for mechanical support of respiratory and cardio-circulatory failure. Excessive systemic inflammatory response is observed during sepsis and after cardiopulmonary bypass (CPB) with similar clinical features. The overwhelming inflammatory response is characterized by highly elevated pro- and anti-inflammatory cytokine levels. The excessive cytokine release during the overwhelming inflammatory response may result in multiple organ damage and failure. During ECMO therapy activation of complement and contact systems occur which may be followed by cytokine release. Controlling excessively increased cytokines may be considered as a valuable treatment option. Hemoadsorption therapy may be used to decrease cytokine levels in case of excessive inflammatory response and due to its unspecific adsorptive characteristics also substances like myoglobin, free hemoglobin or bilirubin. Controlling pro-inflammatory response with hemoadsorption may have positive impact on the endothelial glycocalix and also may be advantageous for maintenance of the vascular barrier function which plays a pivotal role in the development of tissue edema and oxygen mismatch. Hemoadsorption therapy seems to offer a promising new option for the treatment of patients with overwhelming inflammatory response leading to faster hemodynamic and metabolic stabilization finally resulting in preserved organ functions. PMID:29732183

Extracorporeal membrane oxygenation and cytokine adsorption.

PubMed

Datzmann, Thomas; Träger, Karl

2018-03-01

Extracorporeal membrane oxygenation (ECMO) is an increasingly used technology for mechanical support of respiratory and cardio-circulatory failure. Excessive systemic inflammatory response is observed during sepsis and after cardiopulmonary bypass (CPB) with similar clinical features. The overwhelming inflammatory response is characterized by highly elevated pro- and anti-inflammatory cytokine levels. The excessive cytokine release during the overwhelming inflammatory response may result in multiple organ damage and failure. During ECMO therapy activation of complement and contact systems occur which may be followed by cytokine release. Controlling excessively increased cytokines may be considered as a valuable treatment option. Hemoadsorption therapy may be used to decrease cytokine levels in case of excessive inflammatory response and due to its unspecific adsorptive characteristics also substances like myoglobin, free hemoglobin or bilirubin. Controlling pro-inflammatory response with hemoadsorption may have positive impact on the endothelial glycocalix and also may be advantageous for maintenance of the vascular barrier function which plays a pivotal role in the development of tissue edema and oxygen mismatch. Hemoadsorption therapy seems to offer a promising new option for the treatment of patients with overwhelming inflammatory response leading to faster hemodynamic and metabolic stabilization finally resulting in preserved organ functions.

Structural and Kinetic Characterization of the LPS Biosynthetic Enzyme D-alpha,beta-D-heptose-1,7-bisphosphate Phosphatase (GmhB) from Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, P.; Sugiman-Marangos, S; Zhang, K

2010-01-01

Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-{alpha},{beta}-D-Heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis.more » This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting Gram-negative bacterial infection.« less

Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

PubMed

Doerflinger, Sylvie Y; Throop, Andrea L; Herbst-Kralovetz, Melissa M

2014-06-15

Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

PubMed

Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

2018-01-01

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

Biological interaction of living cells with COSAN-based synthetic vesicles

PubMed Central

Tarrés, Màrius; Canetta, Elisabetta; Paul, Eleanor; Forbes, Jordan; Azzouni, Karima; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J.

2015-01-01

Cobaltabisdicarbollide (COSAN) [3,3′-Co(1,2-C2B9H11)2]−, is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes. PMID:25588708

Biological interaction of living cells with COSAN-based synthetic vesicles.

PubMed

Tarrés, Màrius; Canetta, Elisabetta; Paul, Eleanor; Forbes, Jordan; Azzouni, Karima; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

2015-01-15

Cobaltabisdicarbollide (COSAN) [3,3'-Co(1,2-C2B9H11)2](-), is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes.

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina of dorsal root ganglia.

PubMed

Bush, M S; Reid, A R; Allt, G

1991-09-01

Previous investigations of the blood-nerve barrier have correlated the greater permeability of ganglionic endoneurial vessels, compared to those of nerve trunks, with the presence of fenestrations and open intercellular junctions. Recent studies have demonstrated reduced endothelial cell surface charge in blood vessels showing greater permeability. To determine the distribution of anionic sites on the plasma membranes and basal laminae of endothelial cells in dorsal root ganglia, cationic colloidal gold and cationic ferritin were used. Electron microscopy revealed the existence of endothelial microdomains with differing labelling densities. Labelling indicated that caveolar and fenestral diaphragms and basal laminae are highly anionic at physiological pH, luminal plasma membranes and endothelial processes are moderately charged and abluminal plasma membranes are weakly anionic. Tracers did not occur in caveolae or cytoplasmic vesicles. In vitro tracer experiments at pH values of 7.3, 5.0, 3.5 and 2.0 indicated that the anionic charge on the various endothelial domains was contributed by chemical groups with differing pKa values. In summary, the labelling of ganglionic and sciatic nerve vessels was similar except for the heavy labelling of diaphragms in a minority of endoneurial vessels in ganglia. This difference is likely to account in part for the greater permeability of ganglionic endoneurial vessels. The results are discussed with regard to the blood-nerve and -brain barriers and vascular permeability in other tissues and a comparison made between the ultrastructure and anionic microdomains of epi-, peri- and endoneurial vessels of dorsal root ganglia and sciatic nerves.

Cross-over endocytosis of claudins is mediated by interactions via their extracellular loops.

PubMed

Gehne, Nora; Lamik, Agathe; Lehmann, Martin; Haseloff, Reiner F; Andjelkovic, Anuska V; Blasig, Ingolf E

2017-01-01

Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

Nuclear envelope rupture: little holes, big openings.

PubMed

Hatch, Emily M

2018-06-01

The nuclear envelope (NE), which is a critical barrier between the DNA and the cytosol, is capable of extensive dynamic membrane remodeling events in interphase. One of these events, interphase NE rupture and repair, can occur in both normal and disease states and results in the loss of nucleus compartmentalization. NE rupture is not lethal, but new research indicates that it could have broad impacts on genome stability and activate innate immune responses. These observations suggest a new model for how changes in NE structure could be pathogenic in cancer, laminopathies, and autoinflammatory syndromes, and redefine the functions of nucleus compartmentalization. Copyright © 2018 Elsevier Ltd. All rights reserved.

Acting on Actin: Rac and Rho Played by Yersinia.

PubMed

Aepfelbacher, Martin; Wolters, Manuel

2017-01-01

Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.

Development of self-assembled molecular structures on polymeric surfaces and their applications as ultrasonically responsive barrier coatings for on-demand, pulsatile drug delivery

NASA Astrophysics Data System (ADS)

Kwok, Connie Sau-Kuen

Nature in the form of DNA, proteins, and cells has the remarkable ability to interact with its environment by processing biological information through specific molecular recognition at the interface. As such, materials that are capable of triggering an appropriate biological response need to be engineered at the biomaterial surface. Chemically and structurally well-defined self-assembled monolayers (SAMs), biomimetics of the lipid bilayer in cell membranes, have been created and studied mostly on rigid metallic surfaces. This dissertation is motivated by the lack of methods to generate a molecularly designed surface for biomedical polymers and thus provides an enabling technology to engineer a polymeric surface precisely at a molecular and cellular level. To take this innovation one step further, we demonstrated that such self-assembled molecular structure coated on drug-containing polymeric devices could act as a stimulus-responsive barrier for controlled drug delivery. A simple, one-step procedure for generating ordered, crystalline methylene chains on polymeric surfaces via urethane linkages was successfully developed. The self-assemblies and molecular structures of these crystalline methylene chains are comparable to the SAM model surfaces, as evidenced by various surface characterization techniques (XPS, TOF-SIMS, and FTIR-ATR). For the first time, these self-assembled molecular structures are shown to function collectively as an ultrasound-responsive barrier membrane for pulsatile drug delivery, including delivery of low-molecular-weight ciprofloxacin and high-molecular-weight insulin. Encouraging results, based on the insulin-activated deoxyglucose uptakes in adipocytes, indicate that the released insulin remained biologically active. Both chemical and acoustic analyses suggest that the ultrasound-assisted release mechanism is primarily induced by transient cavitation, which causes temporary disruption of the self-assembled overlayer, and thus allows temporal release of the encapsulated drugs. In addition to acoustic energy, self-assembled surfaces experience order-disorder transition and have a transition temperature higher than body temperature if longer alkyl chains (C18) are used. The C18-assembled surface barrier membrane exhibits a relatively superior impermeable coating than the shorter C12 chains. The versatility of derivatizing the terminal groups of the self-assembled molecular structures is illustrated by attaching poly (ethyleneoxide) oligomers to the alkyl chains to minimize nonspecific protein adsorption. This study lays an important foundation for future work in conjugating other biomolecules to develop surface-based diagnostics and biomaterials. With much success, this original research work of forming self-assembled crystalline structures on synthetic materials still allows for numerous opportunities for new applications and possibly even more new discoveries.

Sub-nanometer Atomic Layer Deposition for Spintronics in Magnetic Tunnel Junctions Based on Graphene Spin-Filtering Membranes

PubMed Central

2014-01-01

We report on the successful integration of low-cost, conformal, and versatile atomic layer deposited (ALD) dielectric in Ni–Al2O3–Co magnetic tunnel junctions (MTJs) where the Ni is coated with a spin-filtering graphene membrane. The ALD tunnel barriers, as thin as 0.6 nm, are grown layer-by-layer in a simple, low-vacuum, ozone-based process, which yields high-quality electron-transport barriers as revealed by tunneling characterization. Even under these relaxed conditions, including air exposure of the interfaces, a significant tunnel magnetoresistance is measured highlighting the robustness of the process. The spin-filtering effect of graphene is enhanced, leading to an almost fully inversed spin polarization for the Ni electrode of −42%. This unlocks the potential of ALD for spintronics with conformal, layer-by-layer control of tunnel barriers in magnetic tunnel junctions toward low-cost fabrication and down-scaling of tunnel resistances. PMID:24988469

Placentation and fetal membrane development in the South American coati, Nasua nasua (Mammalia, Carnivora, Procyonidae)

PubMed Central

2014-01-01

Background Placental research in carnivores has concentrated on domestic species, which have zonary, labyrinthine placentas with an endotheliochorial barrier. Although the coati, Nasua nasua, is a widely distributed species in South America, data on the development of the placenta and the fetal membranes in this species are very sparse. Findings Four placentas from mid-gestation to near term were collected from wild individuals and were investigated based on gross morphology, histology, immunohistochemistry and electron microscopy. The available data support the concept that the ancestral condition of placentation in carnivores is phylogenetically characterized by a zonary and labyrinthine placental type with an endotheliochorial fetomaternal barrier, comprising extended epitheliochorial and haemochorial zones, such as hemophagous organs for iron supply and histiotrophe uptake and a yolk sac placenta. Conclusions Because of the foundational mechanisms that lead to the considerable complexity of fetomaternal contact zones in carnivores have not been studied, carnivores are interesting animal models for interhaemal barrier differentiation. PMID:24969476

Decontamination systems information and research program. Quarterly report, April--June 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

This report contains separate reports on the following subtasks: analysis of the Vortec cyclone melting system for remediation of PCB contaminated soils using CFD; drain enhanced soil flushing using prefabricated vertical drains; performance and characteristics evaluation of acrylates as grout barriers; development of standard test protocol barrier design models for desiccation barriers, and for in-situ formed barriers; in-situ bioremediation of chlorinated solvents at Portsmouth Gaseous Diffusion Plant; development of a decision support system and a prototype database for management of the EM50 technology development program; GIS-based infrastructure for site characterization and remediation; treatment of mixed wastes via fluidized bed steammore » reforming; use of centrifugal membrane technology to treat hazardous/radioactive waste; environmental pollution control devices based on novel forms of carbon; development of instrumental methods for analysis of nuclear wastes and environmental materials; production and testing of biosorbents and cleaning solutions for D and D; use of SpinTek centrifugal membrane and sorbents/cleaning solutions for D and D; West Virginia High Tech Consortium Foundation--Environmental support program; small business interaction opportunities; and approach for assessing potential voluntary environmental protection.« less

Clearance of amyloid-β peptide across the choroid plexus in Alzheimer's disease.

PubMed

Alvira-Botero, Ximena; Carro, Eva M

2010-12-01

Aging and several neurodegenerative diseases bring about changes in the anatomy and physiology of the choroid plexus. The identification of specific membrane receptors that bind and internalize extracellular ligands has revolutionized the traditional roles of this tissue. Amyloid beta peptide (Aβ), the major constituent of the amyloid core of senile plaques in patients with Alzheimer's disease (AD) is known to contribute to disease neuropathology and progression. Recent emphasis on comorbidity of AD and a deficient clearance of Aβ across the blood-brain barrier and blood-cerebrospinal fluid barrier have highlighted the importance of brain Aβ clearance in AD. The megalin receptor has also been implicated in the pathogenesis of AD. Faulty Aβ clearance from the brain across the choroid plexus epithelium by megalin appears to mediate focal Aβ accumulation in AD. Patients with AD have reduced levels of megalin at the choroid plexus, which in turn seem to increase brain levels of Aβ through a decreased efflux of brain Aβ. Therapies that increase megalin expression at the choroid plexus could potentially control accumulation of brain Aβ. This review covers in depth the anatomy and function of the choroid plexus, focusing on the brain barrier at the choroid plexus, as it actively participates in Aβ clearance. In addition, we describe the role of the choroid plexus in brain functions, aging and AD, as well as the role of megalin in the process of Aβ clearance. Finally, we present current data on the use of choroid plexus cells to repair the damaged brain.

IFN-γ-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.

PubMed

Kominsky, Douglas J; Campbell, Eric L; Ehrentraut, Stefan F; Wilson, Kelly E; Kelly, Caleb J; Glover, Louise E; Collins, Colm B; Bayless, Amanda J; Saeedi, Bejan; Dobrinskikh, Evgenia; Bowers, Brittelle E; MacManus, Christopher F; Müller, Werner; Colgan, Sean P; Bruder, Dunja

2014-02-01

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

Flexibility contra Stiffness: The Phragmoplast as a Physical Barrier for Beads But Not for Vesicles[OA

PubMed Central

Esseling-Ozdoba, Agnieszka; Kik, Richard A.; van Lammeren, André A.M.; Kleijn, J. Mieke; Emons, Anne Mie C.

2010-01-01

In plant cells, Golgi vesicles are transported to the division plane to fuse with each other, forming the cell plate, the initial membrane-bordered cell wall separating daughter cells. Vesicles, but not organelles, move through the phragmoplast, which consists of two opposing cylinders of microtubules and actin filaments, interlaced with endoplasmic reticulum membrane. To study physical aspects of this transport/inhibition process, we microinjected fluorescent synthetic 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) vesicles and polystyrene beads into Tradescantia virginiana stamen hair cells. The phragmoplast was nonselective for DOPG vesicles of a size up to 150 nm in diameter but was a physical barrier for polystyrene beads having a diameter of 20 and 40 nm and also when beads were coated with the same DOPG membrane. We conclude that stiffness is a parameter for vesicle transit through the phragmoplast and discuss that cytoskeleton configurations can physically block such transit. PMID:19939943

Free Energy Wells and Barriers to Ion Transport Across Membranes

NASA Astrophysics Data System (ADS)

Rempe, Susan

2014-03-01

The flow of ions across cellular membranes is essential to many biological processes. Ion transport is also important in synthetic materials used as battery electrolytes. Transport often involves specific ions and fast conduction. To achieve those properties, ion conduction pathways must solvate specific ions by just the ``right amount.'' The right amount of solvation avoids ion traps due to deep free energy wells, and avoids ion block due to high free energy barriers. Ion channel proteins in cellular membranes demonstrate this subtle balance in solvation of specific ions. Using ab initio molecular simulations, we have interrogated the link between binding site structure and ion solvation free energies in biological ion binding sites. Our results emphasize the surprisingly important role of the environment that surrounds ion-binding sites for fast transport of specific ions. We acknowledge support from Sandia's LDRD program. Sandia National Labs is a multi-program laboratory operated by Sandia Corp., a wholly owned subsidiary of Lockheed Martin Corp., for the US DOE's NNSA under contract DE-AC04-94AL85000.

An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier

PubMed Central

Breslow, David K.; Koslover, Elena F.; Seydel, Federica; Spakowitz, Andrew J.

2013-01-01

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia. PMID:24100294

Development of the first sphingomyelin biomimetic stationary phase for immobilized artificial membrane (IAM) chromatography.

PubMed

Verzele, Dieter; Lynen, Frédéric; De Vrieze, Mike; Wright, Adrian G; Hanna-Brown, Melissa; Sandra, Pat

2012-01-28

A prototype sphingomyelin stationary phase for Immobilized Artificial Membrane (IAM) chromatography was synthesized by an ultra-short, solid-phase inspired methodology, in which an oxidative release monitoring strategy played a vital role. Evaluated in a proof-of-concept model for blood-brain barrier passage, partial least squares regression demonstrated its potential as an in vitro prediction tool.

Comparative exploration of hydrogen sulfide and water transmembrane free energy surfaces via orthogonal space tempering free energy sampling

DOE PAGES

Lv, Chao; Aitchison, Erick W.; Wu, Dongsheng; ...

2015-06-29

Hydrogen sulfide (H 2S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H 2S and its structural analogue, water (H 2O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. Here, as revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is,more » the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H 2S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. Lastly, this study not only explains why H 2S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes.« less

Comparative exploration of hydrogen sulfide and water transmembrane free energy surfaces via orthogonal space tempering free energy sampling.

PubMed

Lv, Chao; Aitchison, Erick W; Wu, Dongsheng; Zheng, Lianqing; Cheng, Xiaolin; Yang, Wei

2016-03-05

Hydrogen sulfide (H2 S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H2 S and its structural analogue, water (H2 O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. As revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is, the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H2 S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2 S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. This study not only explains why H2 S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila.

PubMed

Banerjee, Swati; Pillai, Anilkumar M; Paik, Raehum; Li, Jingjun; Bhat, Manzoor A

2006-03-22

Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. Here, we report the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allowed us to visualize the glial ensheathment of axons. We show that individual or a group of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. We also show that Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and that these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, our data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species.

The Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Fractal

NASA Astrophysics Data System (ADS)

Sadegh, Sanaz; Higgin, Jenny; Mannion, Patrick; Tamkun, Michael; Krapf, Diego

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal. These results present a hierarchical nanoscale picture of the plasma membrane and demonstrate direct interactions between the actin cortex and the cell surface.

Improving Hemocompatibility of Membranes for Extracorporeal Membrane Oxygenators by Grafting Nonthrombogenic Polymer Brushes.

PubMed

Obstals, Fabian; Vorobii, Mariia; Riedel, Tomáš; de Los Santos Pereira, Andres; Bruns, Michael; Singh, Smriti; Rodriguez-Emmenegger, Cesar

2018-03-01

Nonthrombogenic modifications of membranes for extracorporeal membrane oxygenators (ECMOs) are of key interest. The absence of hemocompatibility of these membranes and the need of anticoagulation of patients result in severe and potentially life-threatening complications during ECMO treatment. To address the lack of hemocompatibility of the membrane, surface modifications are developed, which act as barriers to protein adsorption on the membrane and, in this way, prevent activation of the coagulation cascade. The modifications are based on nonionic and zwitterionic polymer brushes grafted directly from poly(4-methyl-1-pentene) (TPX) membranes via single electron transfer-living radical polymerization. Notably, this work introduces the first example of well-controlled surface-initiated radical polymerization of zwitterionic brushes. The antifouling layers markedly increase the recalcification time (a proxy of initiation of coagulation) compared to bare TPX membranes. Furthermore, platelet and leukocyte adhesion is drastically decreased, rendering the ECMO membranes hemocompatible. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanics of Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Powers, Thomas R.

All cells have membranes. The plasma membrane encapsulates the cell's interior, acting as a barrier against the outside world. In cells with nuclei (eukaryotic cells), membranes also form internal compartments (organelles) which carry out specialized tasks, such as protein modification and sorting in the case of the Golgi apparatus, and ATP production in the case of mitochondria. The main components of membranes are lipids and proteins. The proteins can be channels, carriers, receptors, catalysts, signaling molecules, or structural elements, and typically contribute a substantial fraction of the total membrane dry weight. The equilibrium properties of pure lipid membranes are relatively well-understood, and will be the main focus of this article. The framework of elasticity theory and statistical mechanics that we will develop will serve as the foundation for understanding biological phenomena such as the nonequilibrium behavior of membranes laden with ion pumps, the role of membrane elasticity in ion channel gating, and the dynamics of vesicle fission and fusion. Understanding the mechanics of lipid membranes is also important for drug encapsulation and delivery.

Enhancing performance of PEM fuel cells: Using the Au nanoplatelet/Nafion interface to enable CO oxidation under ambient conditions

DOE PAGES

Li, Hongfei; Pan, Cheng; Zhao, Sijia; ...

2016-04-16

We developed a method for fabrication of Au nanoparticle platelets which can be coated onto the Nafion membranes of polymer electrolyte membrane (PEM) fuel cells simply by Langmuir–Blodgett (LB) trough lift off from the air water interface. By incorporating the coated membranes into fuel cells with one membrane electrode assembly (MEA) we enhanced the maximum power output by more than 50% when operated under ambient conditions. An enhancement of more than 200% was observed when 0.1% CO was incorporated into the H 2 input gas stream and minimal enhancement was observed when the PEM fuel cell was operated with 100%more » O 2 gas at the cathode, or when particles were deposited on the electrodes. Density function theory (DFT) calculations were carried out to understand the origin of improved output power. Au NPs with 3-atomic layer in height and 2 nm in size were constructed to model the experimentally synthesized Au NPs. Our results indicated that the Au NPs interacted synergistically with the SO 3 groups, attached at end of Nafion side chains, to reduce the energy barrier for the oxidation of CO occurring at the perimeter of the Au NPs, from 1.292 eV to 0.518 eV, enabling the reaction to occur at T<300 K.« less

Study of polycation effects on erythrocyte agglutination mediated by anti-glycophorins using microscopic image digital analysis

NASA Astrophysics Data System (ADS)

Riquelme, B.; Dumas, D.; Relancio, F.; Fontana, A.; Alessi, A.; Foresto, P.; Grandfils, C.; Stoltz, J.; Valverde, J.

2006-04-01

The aim of this work was to study synthetic polycation effects on erythrocyte agglutination mediated by anti-glycophorin using image digital analysis. Polycations are oligomers or polymers of natural or synthetic origin, which bear a great number of positive charges at pH 7.4. Several of these polycations are nowadays used in clinic for human and veterinary purposes. New applications of polycations to the development of new drug delivery systems are investigated, in order to promote the drug absorption through the gastro-intestinal and blood brain barriers. However, up to now, there are no clear relationships between macromolecular features of polycations (molecular weight, mean charge density, charge repartition, etc.) and their interactions with blood elements (which bear superficial negative charges). The interaction on the red blood cell membrane with synthetic polycations having well-controlled macromolecular features and functionalized with pendent polyethylene glycol segments was investigated. The alterations over stationary and dynamic viscoelastic properties of erythrocyte membranes were analyzed through laser diffractometry. Image digital analysis was used to study erythrocyte agglutination mediated by anti-glycophorin. Results show different reactivities of the polycations on the erythrocyte membrane. These findings could provide more information about the mechanisms of polycation interaction on erythrocyte membranes. We consider that this work could provide useful tools to understand and improve the haemocompatibility of polycations and enlarge their potential in clinic.

Kollidon VA64, a membrane-resealing agent, reduces histopathology and improves functional outcome after controlled cortical impact in mice.

PubMed

Mbye, Lamin H; Keles, Eyup; Tao, Luyang; Zhang, Jimmy; Chung, Joonyong; Larvie, Mykol; Koppula, Rajani; Lo, Eng H; Whalen, Michael J

2012-03-01

Loss of plasma membrane integrity is a feature of acute cellular injury/death in vitro and in vivo. Plasmalemma-resealing agents are protective in acute central nervous system injury models, but their ability to reseal cell membranes in vivo has not been reported. Using a mouse controlled cortical impact (CCI) model, we found that propidium iodide-positive (PI+) cells pulse labeled at 6, 24, or 48 hours maintained a degenerative phenotype and disappeared from the injured brain by 7 days, suggesting that plasmalemma permeability is a biomarker of fatal cellular injury after CCI. Intravenous or intracerebroventricular administration of Kollidon VA64, poloxamer P188, or polyethylene glycol 8000 resealed injured cell membranes in vivo (P<0.05 versus vehicle or poloxamer P407). Kollidon VA64 (1 mmol/L, 500 μL) administered intravenously to mice 1  hour after CCI significantly reduced acute cellular degeneration, chronic brain tissue damage, brain edema, blood-brain barrier damage, and postinjury motor deficits (all P<0.05 versus vehicle). However, VA64 did not rescue pulse-labeled PI+ cells from eventual demise. We conclude that PI permeability within 48 hours of CCI is a biomarker of eventual cell death/loss. Kollidon VA64 reduces secondary damage after CCI by mechanisms other than or in addition to resealing permeable cells.

Periodontal and peri-implant bone regeneration: clinical and histologic observations.

PubMed

Artzi, Z; Zohar, R; Tal, H

1997-02-01

The principle of guided tissue regeneration by barrier membranes to restore lost periodontal tissue around natural teeth has also been used around osseointegrated implants in an attempt to restore alveolar ridge defects. While most periodontal procedures in the literature describe root coverage by mucogingival surgery, which achieves healing through soft tissue attachment, regeneration of denuded root surfaces is performed by guided tissue regeneration using expanded polytetrafluoroethylene barrier membranes and demineralized freeze-dried bone allografts as inductive/conductive materials. In this study the technique is applied in two partially exposed cylindrical hydroxyapatite-coated implants in extraction sites in one patient. Surgical reentry in both sites is presented, with histologic examination revealing new bone formation on the exposed root surface and the hydroxyapatite-coated implants.

Torque Transmission Device at Zero Leakage

NASA Technical Reports Server (NTRS)

Hendricks, R. C.; Mullen, R. L.

2005-01-01

In a few critical applications, mechanical transmission of power by rotation at low speed is required without leakage at an interface. Herein we examine a device that enables torque to be transmitted across a sealed environmental barrier. The barrier represents the restraint membrane through which the torque is transmitted. The power is transferred through elastic deformation of a circular tube into an elliptical cross-section. Rotation of the principle axis of the ellipse at one end results in a commensurate rotation of an elliptical cross section at the other end of the tube. This transfer requires no rigid body rotation of the tube allowing a membrane to seal one end from the other. Both computational and experimental models of the device are presented.

Survival rate of eukaryotic cells following electrophoretic nanoinjection.

PubMed

Simonis, Matthias; Hübner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon

2017-01-25

Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells.

Benzene Adsorption - A Significant Inhibitor for the Hydrogen Oxidation Reaction in Alkaline Conditions

DOE PAGES

Gonzales, Ivana; Chung, Hoon Taek; Kim, Yu Seung

2017-09-25

Slow hydrogen oxidation reaction (HOR) kinetics on Pt under alkaline conditions is a significant technical barrier for the development of high-performance hydroxide exchange membrane fuel cells. Here we report that benzene adsorption on Pt is a major factor responsible for the sluggish HOR. Furthermore, we demonstrate that bimetallic catalysts, such as PtMo/C, PtNi/C, and PtRu/C, can reduce the adsorption of benzene and thereby improve HOR activity. In particular, the HOR voltammogram of PtRu/C in 0.1 M benzyl ammonium showed minimal benzene adsorption. Density functional theory calculations indicate that the adsorption of benzyl ammonium on the bimetallic PtRu is endergonic formore » all four possible orientations of the cation, which explains the significantly better HOR activity observed for the bimetallic catalysts. In conclusion, the new HOR inhibition mechanism described here provides insights for the design of future polymer electrolytes and electrocatalysts for better-performing polymer membrane-based fuel cells.« less

Voltage dependence of a stochastic model of activation of an alpha helical S4 sensor in a K channel membrane

NASA Astrophysics Data System (ADS)

Vaccaro, S. R.

2011-09-01

The voltage dependence of the ionic and gating currents of a K channel is dependent on the activation barriers of a voltage sensor with a potential function which may be derived from the principal electrostatic forces on an S4 segment in an inhomogeneous dielectric medium. By variation of the parameters of a voltage-sensing domain model, consistent with x-ray structures and biophysical data, the lowest frequency of the survival probability of each stationary state derived from a solution of the Smoluchowski equation provides a good fit to the voltage dependence of the slowest time constant of the ionic current in a depolarized membrane, and the gating current exhibits a rising phase that precedes an exponential relaxation. For each depolarizing potential, the calculated time dependence of the survival probabilities of the closed states of an alpha helical S4 sensor are in accord with an empirical model of the ionic and gating currents recorded during the activation process.

Dexpanthenol enhances skin barrier repair and reduces inflammation after sodium lauryl sulphate-induced irritation.

PubMed

Proksch, E; Nissen, H P

2002-12-01

Dexpanthenol-containing creams have been widely used for treatment of lesions (superficial wounds) of the skin and mucous membranes. Dexpanthenol is converted in tissues to pantothenic acid, a component of coenzyme A. Coenzyme A catalyses early steps in the synthesis of fatty acids and sphingolipids which are of crucial importance for stratum corneum lipid bilayers and cell membrane integrity. In the present study, the effects were examined of a dexpanthenol-containing cream on skin barrier repair, stratum corneum hydration, skin roughness, and inflammation after sodium lauryl sulphate (SLS)-induced irritation. Irritation was induced by application of SLS in patch test chambers. The dexpanthenol-contaming cream or the vehicle were applied twice daily and barrier repair, hydration, roughness, and inflammation of the skin were determined by using biophysical methods. Significantly accelerated skin barrier repair was found in treatments with the dexpanthenol-containing cream (verum) compared with vehicle-treated (placebo) or untreated skin. Both verum and placebo showed an increase in stratum corneum hydration, but significantly more so with the dexpanthenol-containing cream. Both creams reduced skin roughness, but again the verum was superior. The dexpanthenol-containing cream significantly reduced skin redness as a sign of inflammation in contrast to the vehicle, which produced no effect. Treatment with a dexpanthenol-containing cream showed significantly enhanced skin barrier repair and stratum corneum hydration, while reducing skin roughness and inflammation.

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

PubMed

Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

2007-01-01

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

Proton transfer in the K-channel analog of B-type Cytochrome c oxidase from Thermus thermophilus.

PubMed

Woelke, Anna Lena; Wagner, Anke; Galstyan, Gegham; Meyer, Tim; Knapp, Ernst-Walter

2014-11-04

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers.

Proton Transfer in the K-Channel Analog of B-Type Cytochrome c Oxidase from Thermus thermophilus

PubMed Central

Woelke, Anna Lena; Wagner, Anke; Galstyan, Gegham; Meyer, Tim; Knapp, Ernst-Walter

2014-01-01

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers. PMID:25418102

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

PubMed

Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

2017-03-01

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Interaction of partially denatured insulin with a DSPC floating lipid bilayer.

PubMed

Dennison, A J C; Jones, R A L; Staniforth, R A; Parnell, A J

2016-01-21

The carefully controlled permeability of cellular membranes to biological molecules is key to life. In degenerative diseases associated with protein misfolding and aggregation, protein molecules or their aggregates are believed to permeate these barriers and threaten membrane integrity. We used neutron reflectivity to study the interaction of insulin, a model amyloidogenic protein, with a DSPC floating lipid bilayer. Structural changes consistent with protein partitioning to the membrane interior and adsorption to a gel phase model lipid bilayer were observed under conditions where the native fold of the protein is significantly destabilised. We propose that the perturbation of the membrane by misfolded proteins involves long term occupation of the membrane by these proteins, rather than transient perforation events.

Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites

PubMed Central

Li, Yinfeng; Yuan, Hongyan; von dem Bussche, Annette; Creighton, Megan; Hurt, Robert H.; Kane, Agnes B.; Gao, Huajian

2013-01-01

Understanding and controlling the interaction of graphene-based materials with cell membranes is key to the development of graphene-enabled biomedical technologies and to the management of graphene health and safety issues. Very little is known about the fundamental behavior of cell membranes exposed to ultrathin 2D synthetic materials. Here we investigate the interactions of graphene and few-layer graphene (FLG) microsheets with three cell types and with model lipid bilayers by combining coarse-grained molecular dynamics (MD), all-atom MD, analytical modeling, confocal fluorescence imaging, and electron microscopic imaging. The imaging experiments show edge-first uptake and complete internalization for a range of FLG samples of 0.5- to 10-μm lateral dimension. In contrast, the simulations show large energy barriers relative to kBT for membrane penetration by model graphene or FLG microsheets of similar size. More detailed simulations resolve this paradox by showing that entry is initiated at corners or asperities that are abundant along the irregular edges of fabricated graphene materials. Local piercing by these sharp protrusions initiates membrane propagation along the extended graphene edge and thus avoids the high energy barrier calculated in simple idealized MD simulations. We propose that this mechanism allows cellular uptake of even large multilayer sheets of micrometer-scale lateral dimension, which is consistent with our multimodal bioimaging results for primary human keratinocytes, human lung epithelial cells, and murine macrophages. PMID:23840061

Fabrication of semi-aromatic polyamide/spherical mesoporous silica nanocomposite reverse osmosis membrane with superior permeability

NASA Astrophysics Data System (ADS)

Li, Qiang; Yu, Hui; Wu, Feiyang; Song, Jie; Pan, Xianhui; Zhang, Meng

2016-02-01

Semi-aromatic polyamide (SAP)/spherical mesoporous silica nanocomposite reverse osmosis (RO) membrane was successfully fabricated using m-phenylene diamine aqueous solution and cyclohexane-1,3,5-tricarbonyl chloride/mesoporous-silica-sphere (MSS) organic solution as main raw materials. The experimental suggests that the microstructures and surface features are significantly different from those of the contrast samples (the full- and semi-aromatic polyamide membranes), including the surface morphology, polymer framework structure, surface charge density, hydrophilicity, and the thickness of barrier layer. It was observed that many MSSs with ca. 1.5 nm of pore size are evenly embedded on the surface of the fabricated SAP/MSS RO membrane. Furthermore, the separation performance testing results indicate that the permeabilities range from 62.53 to 72.73 L/m2 h with the increase of the introduced MSSs from 0.02 to 0.08 w/v % under 1.5 MPa operating pressure and 2000 mg/L NaCl solution, which is obviously better than the contrast samples. Simultaneously, their salt rejections can be still maintained at a comparable level (94.78-91.46%). The excellent separation performance of the nanocomposite RO membrane is closely related to the higher-freedom-degree semi-aromatic framework, the incorporation of MSSs, the improved surface hydrophilicity, the thinner barrier layer, and the enhanced surface negative charge density.

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

PubMed

Palestini, Paola; Calvi, Chiara; Conforti, Elena; Daffara, Rossella; Botto, Laura; Miserocchi, Giuseppe

2003-10-01

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

A Comprehensive Biophysical Model of Ion and Water Transport in Plant Roots. I. Clarifying the Roles of Endodermal Barriers in the Salt Stress Response

PubMed Central

Foster, Kylie J.; Miklavcic, Stanley J.

2017-01-01

In this paper, we present a detailed and comprehensive mathematical model of active and passive ion and water transport in plant roots. Two key features are the explicit consideration of the separate, but interconnected, apoplastic, and symplastic transport pathways for ions and water, and the inclusion of both active and passive ion transport mechanisms. The model is used to investigate the respective roles of the endodermal Casparian strip and suberin lamellae in the salt stress response of plant roots. While it is thought that these barriers influence different transport pathways, it has proven difficult to distinguish their separate functions experimentally. In particular, the specific role of the suberin lamellae has been unclear. A key finding based on our simulations was that the Casparian strip is essential in preventing excessive uptake of Na+ into the plant via apoplastic bypass, with a barrier efficiency that is reflected by a sharp gradient in the steady-state radial distribution of apoplastic Na+ across the barrier. Even more significantly, this function cannot be replaced by the action of membrane transporters. The simulations also demonstrated that the positive effect of the Casparian strip of controlling Na+ uptake, was somewhat offset by its contribution to the osmotic stress component: a more effective barrier increased the detrimental osmotic stress effect. In contrast, the suberin lamellae were found to play a relatively minor, even non-essential, role in the overall response to salt stress, with the presence of the suberin lamellae resulting in only a slight reduction in Na+ uptake. However, perhaps more significantly, the simulations identified a possible role of suberin lamellae in reducing plant energy requirements by acting as a physical barrier to preventing the passive leakage of Na+ into endodermal cells. The model results suggest that more and particular experimental attention should be paid to the properties of the Casparian strip when assessing the salt tolerance of different plant varieties and species. Indeed, the Casparian strip appears to be a more promising target for plant breeding and plant genetic engineering efforts than the suberin lamellae for the goal of improving salt tolerance. PMID:28804493

Oxygen reduction on a Pt(111) catalyst in HT-PEM fuel cells by density functional theory

NASA Astrophysics Data System (ADS)

Sun, Hong; Li, Jie; Almheiri, Saif; Xiao, Jianyu

2017-08-01

The oxygen reduction reaction plays an important role in the performance of high-temperature proton exchange membrane (HT-PEM) fuel cells. In this study, a molecular dynamics model, which is based on the density functional theory and couples the system's energy, the exchange-correlation energy functional, the charge density distribution function, and the simplified Kohn-Sham equation, was developed to simulate the oxygen reduction reaction on a Pt(111) surface. Additionally, an electrochemical reaction system on the basis of a four-electron reaction mechanism was also developed for this simulation. The reaction path of the oxygen reduction reaction, the product structure of each reaction step and the system's energy were simulated. It is found that the first step reaction of the first hydrogen ion with the oxygen molecule is the controlling step of the overall reaction. Increasing the operating temperature speeds up the first step reaction rate and slightly decreases its reaction energy barrier. Our results provide insight into the working principles of HT-PEM fuel cells.

Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection.

PubMed

Hu, Xiaolong; Zhu, Min; Liang, Zi; Kumar, Dhiraj; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

2017-04-01

The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

Stability and dynamics of membrane-spanning DNA nanopores

NASA Astrophysics Data System (ADS)

Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

2017-03-01

Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine.

Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids

PubMed Central

1993-01-01

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein- phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter. PMID:8416991

Surface charge- and space-dependent transport of proteins in crowded environments of nanotailored posts.

PubMed

Choi, Chang Kyoung; Fowlkes, Jason D; Retterer, Scott T; Siuti, Piro; Iyer, Sukanya; Doktycz, Mitchel J

2010-06-22

The reaction and diffusion of molecules across barriers and through crowded environments is integral to biological system function and to separation technologies. Ordered, microfabricated post arrays are a promising route to creating synthetic barriers with controlled chemical and physical characteristics. They can be used to create crowded environments, to mimic aspects of cellular membranes, and to serve as engineered replacements of polymer-based separation media. Here, the translational diffusion of fluorescein isothiocyante and various forms of green fluorescent protein (GFP), including "supercharged" variants, are examined in a silicon-based post array environment. The technique of fluorescence recovery after photobleaching (FRAP) is combined with analytical approximations and numerical simulations to assess the relative effects of reaction and diffusion on molecular transport, respectively. FRAP experiments were conducted for 64 different cases where the molecular species, the density of the posts, and the chemical surface charge of the posts were varied. In all cases, the dense packing of the posts hindered the diffusive transport of the fluorescent species. The supercharged GFPs strongly interacted with oppositely charged surfaces. With similar molecular and surface charges, transport is primarily limited by hindered diffusion. For conventional, enhanced GFP in a positively charged surface environment, transport was limited by the coupled action of hindered diffusion and surface interaction with the posts. Quantification of the size-, space-, time-, and charge-dependent translational diffusion in the post array environments can provide insight into natural processes and guide the design and development of selective membrane systems.

Differentiation-dependent rearrangements of actin filaments and microtubules hinder apical endocytosis in urothelial cells.

PubMed

Tratnjek, Larisa; Romih, Rok; Kreft, Mateja Erdani

2017-08-01

During differentiation, superficial urothelial cells (UCs) of the urinary bladder form the apical surface, which is almost entirely covered by urothelial plaques containing densely packed uroplakin particles. These urothelial plaques are the main structural components of the blood-urine permeability barrier in the urinary bladder. We have shown previously that endocytosis from the apical plasma membrane decreases during urothelial cell differentiation. Here, we investigated the role of actin filament and microtubule rearrangements in apical endocytosis of differentiating UCs cells using hyperplastic and normoplastic porcine urothelial models. Partially differentiated normal porcine UCs contained actin filaments in the subapical cytoplasm, while microtubules had a net-like appearance. In highly differentiated UCs, actin filaments mostly disappeared from the subapical cytoplasm and microtubules remained as a thin layer close to the apical plasma membrane. Inhibition of actin filament formation with cytochalasin-D in partially differentiated UCs caused a decrease in apical endocytosis. Depolymerisation of microtubules with nocodazole did not prevent endocytosis of the endocytotic marker WGA into the subapical cytoplasm; however, it abolished WGA transport to endolysosomal compartments in the central cytoplasm. Cytochalasin-D or nocodazole treatment did not significantly change apical endocytosis in highly differentiated UCs. In conclusion, we showed that the physiological differentiation-dependent or chemically induced redistribution and reorganization of actin filaments and microtubules impair apical endocytosis in UCs. Importantly, reduced apical endocytosis due to cytoskeletal rearrangements in highly differentiated UCs, together with the formation of rigid urothelial plaques, reinforces the barrier function of the urothelium.

Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance.

PubMed

Lange, K; Gartzke, J

2001-08-01

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.

Hemodiafiltration: Technical and Clinical Issues.

PubMed

Ronco, Claudio

2015-01-01

Hemodiafiltration (HDF) seems to represent the gold standard in the field of replacement of renal function by dialysis. High convective fluxes have been correlated with better clinical outcomes. Sometimes, however, there are technical barriers to the achievement of high blood flows adequate to perform effective convective therapies. In spite of optimized procedures, the progressive increase in transmembrane pressure (TMP), the blood viscosity due to hemoconcentration and blood path resistance sometimes becomes inevitable. We propose two possible solutions that can be operated automatically via specific software in the dialysis machine: predilution on demand and backflush on demand. Predilution on demand consists in an automatic feedback of the machine, diverting part of the filtered dialysate into a predilution mode with an infusion of 200 ml in 30 s while the ultrafiltration pump stops. This produces a sudden hemodilution with a return of the parameters to acceptable values. The performance of the filter improves, and the pressure alterations are mitigated. Backflush on demand consists in an automatic feedback of the machine triggered by the TMP control, producing a positive pressure in the dialysate compartment due to a stop of filtration and rapid infusion of at least 100 ml of ultrapure dialysate into the hollow fiber. This not only produces a significant hemodilution, but also backflushes the membrane pores detaching protein layers and improving membrane permeability. These are two examples of how technology will permit to overcome technical barriers to a widespread diffusion of HDF and adequate convective dose delivery. © 2015 S. Karger AG, Basel.

Ion Transport through Membrane-Spanning Nanopores Studied by Molecular Dynamics Simulations and Continuum Electrostatics Calculations

PubMed Central

Peter, Christine; Hummer, Gerhard

2005-01-01

Narrow hydrophobic regions are a common feature of biological channels, with possible roles in ion-channel gating. We study the principles that govern ion transport through narrow hydrophobic membrane pores by molecular dynamics simulation of model membranes formed of hexagonally packed carbon nanotubes. We focus on the factors that determine the energetics of ion translocation through such nonpolar nanopores and compare the resulting free-energy barriers for pores with different diameters corresponding to the gating regions in closed and open forms of potassium channels. Our model system also allows us to compare the results from molecular dynamics simulations directly to continuum electrostatics calculations. Both simulations and continuum calculations show that subnanometer wide pores pose a huge free-energy barrier for ions, but a small increase in the pore diameter to ∼1 nm nearly eliminates that barrier. We also find that in those wider channels the ion mobility is comparable to that in the bulk phase. By calculating local electrostatic potentials, we show that the long range Coulomb interactions of ions are strongly screened in the wide water-filled channels. Whereas continuum calculations capture the overall energetics reasonably well, the local water structure, which is not accounted for in this model, leads to interesting effects such as the preference of hydrated ions to move along the pore wall rather than through the center of the pore. PMID:16006629