Monitoring Physiological Variables with Membrane Probes

NASA Technical Reports Server (NTRS)

Janle, Elsa M.

1997-01-01

This project has demonstrated the possibility of using membrane probes in rodents to monitor physiological variables for extended periods of time. The utility of these probes in physiological studies of microgravity has been demonstrated. The feasibility of developing on-line sensors has also been demonstrated and allows for the possibility of developing real-time automated monitoring systems which can be used in ground-base physiological research as well as in research and medical monitoring in space. In addition to space applications these techniques can be extended to medical monitoring in critical care situations on earth as well as facilitating research in many human and animal diseases.

Molecular Probes: An Innovative Technology for Monitoring Membrane Processes

NASA Astrophysics Data System (ADS)

Santoro, Sergio

The ultimate objective of this study is to use molecular probes as an innovative and alternative technology contributing to the advance of membrane science by monitoring membrane processes in-situ, on-line and at sub-micron scale. An optical sensor for oxygen sensing was developed by the immobilization of tris (1,10-phenanthroline) ruthenium (II) (Ru(phen)3) in a dense polymeric membrane made of polystyrene (PS) or Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The emission of the probe was quenched by both the temperature and by the oxygen. Moreover, the oxygen sensitivity was affected by the oxygen permeability of the membrane. The evaluation of the oxygen concentration is prone to errors since the emission of a single probe depends on several parameters (i.e. optical path, source intensity). The correction of these artefacts was obtained by the immobilization of a second luminescent molecule non-sensitive to the oxygen, Coumarin. The potential of the luminescent ratiometric sensor for the non-invasive monitoring of oxygen in food packaging using polymeric films with different oxygen permeability was evaluated. Emphasis was given to the efficiency of the optical sensor for the on-line, in-situ and non invasive monitoring of the oxygen by comparing the experimental data with a model which takes into account the oxygen permeability of the packaging materials evaluated independently. A nano-thermometer based on silica nano-particles doped with Ru(phen)3 was developed. A systematic study shows how it is possible to control the properties of the nano-particles as well as their temperature sensitivity. The nano-thermometer was immobilized on a membrane surface by dip-coating providing information about the temperature on the membrane surface. Hydrophobic porous membrane made of Poly(vinylidene fluoride) was prepared via electrospinning and employed in a direct contact membrane distillation process. Using a designed membrane module and a membrane doped with Ru(phen)3 the on-line mapping of the temperature on the membrane's surface was evaluated. None None None None

Monitoring Membrane Hydration with 2-(Dimethylamino)-6-Acylnaphtalenes Fluorescent Probes.

PubMed

Bagatolli, Luis A

2015-01-01

A family of polarity sensitive fluorescent probes (2-(dimethylamino)-6-acylnaphtalenes, i.e. LAURDAN, PRODAN, ACDAN) was introduced by Gregorio Weber in 1979, with the aim to monitor solvent relaxation phenomena on protein matrices. In the following years, however, PRODAN and particularly LAURDAN, were used to study membrane lateral structure and associated dynamics. Once incorporated into membranes, the (nanosecond) fluorescent decay of these probes is strongly affected by changes in the local polarity and relaxation dynamics of restricted water molecules existing at the membrane/water interface. For instance, when glycerophospholipid containing membranes undertake a solid ordered (gel) to liquid disordered phase transition the fluorescence emission maximum of these probes shift ~ 50 nm with a significant change in their fluorescence lifetime. Furthermore, the fluorescence parameters of LAURDAN and PRODAN are exquisitely sensitive to cholesterol effects, allowing interpretations that correlate changes in membrane packing with membrane hydration. Different membrane model systems as well as innate biological membranes have been studied with this family of probes allowing interesting comparative studies. This chapter presents a short historical overview about these fluorescent reporters, discusses on different models proposed to explain their sensitivity to membrane hydration, and includes relevant examples from experiments performed in artificial and biological membranes.

Rapid labeling of intracellular His-tagged proteins in living cells.

PubMed

Lai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, Hongzhe

2015-03-10

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Luminescence Probe Studies of Ionomers. I. Steady-State Measurements from Nafion Membrane.

DTIC Science & Technology

1985-02-03

Bu4N+ was monitored by titrating the released protons with NaOH. Bu4N+ uptake by Na+ membranes was assumed to be the same as uptake by acidic form...spaced excited states (21). Because of their solvent sensitivity, 1,5-DNS derivatives may also be used as polarity probes (20). The acid -base... acid . The basic (unprotonated) form of the DNS derivative used here (DA+ ) shows *absorption maxima at ca. 325 and 245 nm (aqueous solution, pH 6.8

DNA probe for monitoring dynamic and transient molecular encounters on live cell membranes

PubMed Central

You, Mingxu; Lyu, Yifan; Han, Da; Qiu, Liping; Liu, Qiaoling; Chen, Tao; Wu, Cuichen Sam; Peng, Lu; Zhang, Liqin; Bao, Gang; Tan, Weihong

2017-01-01

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, such as motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within different lipid domains. PMID:28319616

Composite sensor membrane

DOEpatents

Majumdar, Arun [Orinda, CA; Satyanarayana, Srinath [Berkeley, CA; Yue, Min [Albany, CA

2008-03-18

A sensor may include a membrane to deflect in response to a change in surface stress, where a layer on the membrane is to couple one or more probe molecules with the membrane. The membrane may deflect when a target molecule reacts with one or more probe molecules.

A Cu-free clickable fluorescent probe for intracellular targeting of small biomolecules.

PubMed

Yamagishi, Kento; Sawaki, Kazuaki; Murata, Atsushi; Takeoka, Shinji

2015-05-07

We synthesized a novel cyclooctyne-based clickable fluorescent probe with versatile properties such as high cell-membrane permeability and free diffusibility in the cell. Our probe "FC-DBCO" was conjugated to an azide-modified mannose via a Cu-free click reaction in living HeLa cells and displayed intracellular specific fluorescence imaging with low background signals.

Long Time-lapse Nanoscopy with Spontaneously Blinking Membrane Probes

PubMed Central

Takakura, Hideo; Zhang, Yongdeng; Erdmann, Roman S.; Thompson, Alexander D.; Lin, Yu; McNellis, Brian; Rivera-Molina, Felix; Uno, Shin-nosuke; Kamiya, Mako; Urano, Yasuteru; Rothman, James E.; Bewersdorf, Joerg; Schepartz, Alanna; Toomre, Derek

2017-01-01

Long time-lapse, diffraction-unlimited super-resolution imaging of cellular structures and organelles in living cells is highly challenging, as it requires dense labeling, bright, highly photostable dyes, and non-toxic conditions. We developed a set of high-density, environment-sensitive (HIDE) membrane probes based on HMSiR that assemble in situ and enable long time-lapse, live cell nanoscopy of discrete cellular structures and organelles with high spatio-temporal resolution. HIDE-enabled nanoscopy movies are up to 50x longer than movies obtained with labeled proteins, reveal the 2D dynamics of the mitochondria, plasma membrane, and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum in living cells. These new HIDE probes also facilitate the acquisition of live cell, two-color, super-resolution images, greatly expanding the utility of nanoscopy to visualize processes and structures in living cells. PMID:28671662

Quantum dots-based probes conjugated to Annexin V for photostable apoptosis detection and imaging

NASA Astrophysics Data System (ADS)

Le Gac, Séverine; Vermes, Istvan; van den Berg, Albert

2008-02-01

Quantum dots (Qdots) are nanoparticles exhibiting fluorescent properties that are widely applied for cell staining. We present here the development of quantum dots for specific targeting of apoptotic cells, for both apoptosis detection and staining of apoptotic "living" cells. These Qdots are functionalized with Annexin V, a 35-kDa protein that specifically interacts with the membrane of apoptotic cells: Annexin V recognizes and binds to phosphatidylserine (PS) moieties which are present on the outer membrane of apoptotic cells and not on this of healthy or necrotic cells. By using Annexin V, our Qdots probes are made specific for apoptotic cells. For that purpose, Qdots Streptavidin Conjugates are coupled to biotinylated Annexin V. Staining of apoptotic cells was checked using fluorescence and confocal microscopy techniques on nonfixed cells. It is shown here that Qdots are insensitive to bleaching after prolonged and frequent exposure as opposed to organic dyes and this makes them excellent candidates for time-lapse imaging purposes. We illustrate the application of our Qdots-based probes to continuously follow fast changes occurring on the membrane of apoptotic cells.

Fluorescent-protein-based probes: general principles and practices.

PubMed

Ai, Hui-Wang

2015-01-01

An important application of fluorescent proteins is to derive genetically encoded fluorescent probes that can actively respond to cellular dynamics such as pH change, redox signaling, calcium oscillation, enzyme activities, and membrane potential. Despite the large diverse group of fluorescent-protein-based probes, a few basic principles have been established and are shared by most of these probes. In this article, the focus is on these general principles and strategies that guide the development of fluorescent-protein-based probes. A few examples are provided in each category to illustrate the corresponding principles. Since these principles are quite straightforward, others may adapt them to create fluorescent probes for their own interest. Hopefully, the development of the ever-growing family of fluorescent-protein-based probes will no longer be limited to a small number of laboratories specialized in senor development, leading to the situation that biological studies will be bettered assisted by genetically encoded sensors.

Rapid labeling of intracellular His-tagged proteins in living cells

PubMed Central

Lai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A.; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, Hongzhe

2015-01-01

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells. PMID:25713372

Indentation of Graphene-Covered Atomic Force Microscopy Probe Across a Lipid Bilayer Membrane: Effect of Tip Shape, Size, and Surface Hydrophobicity.

PubMed

Lv, Kang; Li, Yinfeng

2018-06-21

Understanding the interaction of graphene with cell membranes is crucial to the development of graphene-based biological applications and the management of graphene safety issues. To help reveal the key factors controlling the interaction between graphene and cell membranes, here we adopt the dissipative particle dynamics method to analyze the evolution of interaction force and free energy as the graphene-covered atomic force microscopy (AFM) probe indents across a lipid bilayer. The simulation results show that the graphene-covered AFM probe can cause severe deformation of the cell membrane which drives the lipid molecule to adsorb and diffuse at the surface of graphene. The breakthrough force and free energy are calculated to study the effects of the tip shape, size, and surface hydrophobicity on the piercing behaviors of graphene-covered AFM. In addition, the deformation of cell membrane can decrease the dependency of the breakthrough force on the tip shape. The analysis of surface functionalization suggests that the horizontal patterns on graphene can change the preferred orientation in the penetration process, but the vertical patterns on graphene may disrupt the cell membrane. What's more, the bending stiffness of graphene has little influence on the penetration process as graphene pierces into the cell membrane. These results provide useful guidelines for the molecular design of graphene materials with controllable cell penetrability.

Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes.

PubMed

Roland, Felicia M; Peck, Evan M; Rice, Douglas R; Smith, Bradley D

2017-04-19

A new self-assembly process known as Synthavidin (synthetic avidin) technology was used to prepare targeted probes for near-infrared fluorescence imaging of anionic membranes and cell surfaces, a hallmark of many different types of disease. The probes were preassembled by threading a tetralactam macrocycle with six appended zinc-dipicolylamine (ZnDPA) targeting units onto a linear scaffold with one or two squaraine docking stations to produce hexavalent or dodecavalent fluorescent probes. A series of liposome titration experiments showed that multivalency promoted stronger membrane binding by the dodecavalent probe. In addition, the dodecavalent probe exhibited turn-on fluorescence due to probe unfolding during fluorescence microscopy at the membrane surface. However, the dodecavalent probe also had a higher tendency to self-aggregate after membrane binding, leading to probe self-quenching under certain conditions. This self-quenching effect was apparent during fluorescence microscopy experiments that recorded low fluorescence intensity from anionic dead and dying mammalian cells that were saturated with the dodecavalent probe. Conversely, probe self-quenching was not a factor with anionic microbial surfaces, where there was intense fluorescence staining by the dodecavalent probe. A successful set of rat tumor imaging experiments confirmed that the preassembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of preassembled fluorescent probes for multivalent targeting, but fluorescence imaging performance depends on the specific physical attributes of the biomarker target, such as the spatial distance between different copies of the biomarker and the propensity of the probe-biomarker complex to self-aggregate.

Designing topological defects in 2D materials using scanning probe microscopy and a self-healing mechanism: a density functional-based molecular dynamics study

NASA Astrophysics Data System (ADS)

Popov, Igor; Đurišić, Ivana; Belić, Milivoj R.

2017-12-01

Engineering of materials at the atomic level is one of the most important aims of nanotechnology. The unprecedented ability of scanning probe microscopy to address individual atoms opened up the possibilities for nanomanipulation and nanolitography of surfaces and later on of two-dimensional materials. While the state-of-the-art scanning probe lithographic methods include, primarily, adsorption, desorption and repositioning of adatoms and molecules on substrates or tailoring nanoribbons by etching of trenches, the precise modification of the intrinsic atomic structure of materials is yet to be advanced. Here we introduce a new concept, scanning probe microscopy with a rotating tip, for engineering of the atomic structure of membranes based on two-dimensional materials. In order to indicate the viability of the concept, we present our theoretical research, which includes atomistic modeling, molecular dynamics simulations, Fourier analysis and electronic transport calculations. While stretching can be employed for fabrication of atomic chains only, our comprehensive molecular dynamics simulations indicate that nanomanipulation by scanning probe microscopy with a rotating tip is capable of assembling a wide range of topological defects in two-dimensional materials in a rather controllable and reproducible manner. We analyze two possibilities. In the first case the probe tip is retracted from the membrane while in the second case the tip is released beneath the membrane allowing graphene to freely relax and self-heal the pore made by the tip. The former approach with the tip rotation can be achieved experimentally by rotation of the sample, which is equivalent to rotation of the tip, whereas irradiation of the membrane by nanoclusters can be utilized for the latter approach. The latter one has the potential to yield a yet richer diversity of topological defects on account of a lesser determinacy. If successfully realized experimentally the concept proposed here could be an important step toward controllable nanostructuring of two-dimensional materials.

Designing topological defects in 2D materials using scanning probe microscopy and a self-healing mechanism: a density functional-based molecular dynamics study.

PubMed

Popov, Igor; Đurišić, Ivana; Belić, Milivoj R

2017-12-08

Engineering of materials at the atomic level is one of the most important aims of nanotechnology. The unprecedented ability of scanning probe microscopy to address individual atoms opened up the possibilities for nanomanipulation and nanolitography of surfaces and later on of two-dimensional materials. While the state-of-the-art scanning probe lithographic methods include, primarily, adsorption, desorption and repositioning of adatoms and molecules on substrates or tailoring nanoribbons by etching of trenches, the precise modification of the intrinsic atomic structure of materials is yet to be advanced. Here we introduce a new concept, scanning probe microscopy with a rotating tip, for engineering of the atomic structure of membranes based on two-dimensional materials. In order to indicate the viability of the concept, we present our theoretical research, which includes atomistic modeling, molecular dynamics simulations, Fourier analysis and electronic transport calculations. While stretching can be employed for fabrication of atomic chains only, our comprehensive molecular dynamics simulations indicate that nanomanipulation by scanning probe microscopy with a rotating tip is capable of assembling a wide range of topological defects in two-dimensional materials in a rather controllable and reproducible manner. We analyze two possibilities. In the first case the probe tip is retracted from the membrane while in the second case the tip is released beneath the membrane allowing graphene to freely relax and self-heal the pore made by the tip. The former approach with the tip rotation can be achieved experimentally by rotation of the sample, which is equivalent to rotation of the tip, whereas irradiation of the membrane by nanoclusters can be utilized for the latter approach. The latter one has the potential to yield a yet richer diversity of topological defects on account of a lesser determinacy. If successfully realized experimentally the concept proposed here could be an important step toward controllable nanostructuring of two-dimensional materials.

Light on fluorescent lipids in rafts: a lesson from model membranes.

PubMed

Kahya, Nicoletta

2010-09-15

Tracking fluorescent lipids in cellular membranes has been applied for decades to shed light on membrane trafficking, sorting, endocytosis and exocytosis, viral entry, and to understand the functional relevance of membrane heterogeneity, phase separation and lipid rafts. However, fluorescent probes may display different organizing behaviour from their corresponding endogenous lipids. A full characterization of these probes is therefore required for proper interpretation of fluorescence microscopy data in complex membrane systems. Model membrane studies provide essential clues that guide us to design and interpret our experiments, help us to avoid pitfalls and resolve artefacts in complex cellular environments. In the present issue of the Biochemical Journal, Juhasz, Davis and Sharom demonstrate the importance of testing lipid probes systematically in heterogeneous model membranes of specific composition and well-defined thermodynamic properties. The phase-partitioning behaviour of fluorescent probes, alone and/or in combination, cannot simply be assumed, but has to be fully characterized.

Novel benzanthrone probes for membrane and protein studies

NASA Astrophysics Data System (ADS)

Ryzhova, Olga; Vus, Kateryna; Trusova, Valeriya; Kirilova, Elena; Kirilov, Georgiy; Gorbenko, Galyna; Kinnunen, Paavo

2016-09-01

The applicability of a series of novel benzanthrone dyes to monitoring the changes in physicochemical properties of lipid bilayer and to differentiating between the native and aggregated protein states has been evaluated. Based on the quantitative parameters of the dye-membrane and dye-protein binding derived from the fluorimetric titration data, the most prospective membrane probes and amyloid tracers have been selected from the group of examined compounds. Analysis of the red edge excitation shifts of the membrane- and amyloid-bound dyes provided information on the properties of benzanthrone binding sites within the lipid and protein matrixes. To understand how amyloid specificity of benzanthrones correlates with their structure, quantitative structure activity relationship (QSAR) analysis was performed involving a range of quantum chemical molecular descriptors. A statistically significant model was obtained for predicting the sensitivity of novel benzanthrone dyes to amyloid fibrils.

Probing the Interaction of Dielectric Nanoparticles with Supported Lipid Membrane Coatings on Nanoplasmonic Arrays

PubMed Central

Ferhan, Abdul Rahim; Ma, Gamaliel Junren; Jackman, Joshua A.; Sut, Tun Naw; Park, Jae Hyeon; Cho, Nam-Joon

2017-01-01

The integration of supported lipid membranes with surface-based nanoplasmonic arrays provides a powerful sensing approach to investigate biointerfacial phenomena at membrane interfaces. While a growing number of lipid vesicles, protein, and nucleic acid systems have been explored with nanoplasmonic sensors, there has been only very limited investigation of the interactions between solution-phase nanomaterials and supported lipid membranes. Herein, we established a surface-based localized surface plasmon resonance (LSPR) sensing platform for probing the interaction of dielectric nanoparticles with supported lipid bilayer (SLB)-coated, plasmonic nanodisk arrays. A key emphasis was placed on controlling membrane functionality by tuning the membrane surface charge vis-à-vis lipid composition. The optical sensing properties of the bare and SLB-coated sensor surfaces were quantitatively compared, and provided an experimental approach to evaluate nanoparticle–membrane interactions across different SLB platforms. While the interaction of negatively-charged silica nanoparticles (SiNPs) with a zwitterionic SLB resulted in monotonic adsorption, a stronger interaction with a positively-charged SLB resulted in adsorption and lipid transfer from the SLB to the SiNP surface, in turn influencing the LSPR measurement responses based on the changing spatial proximity of transferred lipids relative to the sensor surface. Precoating SiNPs with bovine serum albumin (BSA) suppressed lipid transfer, resulting in monotonic adsorption onto both zwitterionic and positively-charged SLBs. Collectively, our findings contribute a quantitative understanding of how supported lipid membrane coatings influence the sensing performance of nanoplasmonic arrays, and demonstrate how the high surface sensitivity of nanoplasmonic sensors is well-suited for detecting the complex interactions between nanoparticles and lipid membranes. PMID:28644423

Location of fluorescent probes (2'-hydroxy derivatives of 2,5-diaryl-1,3-oxazole) in lipid membrane studied by fluorescence spectroscopy and molecular dynamics simulation.

PubMed

Posokhov, Yevgen; Kyrychenko, Alexander

2018-04-01

2'-Hydroxy derivatives of 2,5-diaryl-1,3-oxazole are known as environment-sensitive ratiometric excited-state intramolecular proton transfer (ESIPT) fluorescent probes, which are used to monitor physicochemical properties of lipid membranes. However, because of their heterogeneous membrane distribution, accurate experimental determination of the probe position is difficult. To estimate the location of the ESIPT probes in lipid membranes we have performed fluorescence measurements and molecular dynamics (MD) simulations. In the series composed of 2-(2'-hydroxy-phenyl)-5-phenyl-1,3-oxazole (1), 2-(2'-hydroxy-phenyl)-5-(4'-biphenyl)-1,3-oxazole (2), and 2-(2'-hydroxy-phenyl)-phenanthro[9,10-d]-1,3-oxazole (3), the structure of the ESIPT-moiety of 2-(2'-hydroxy-phenyl)-oxazole was varied by either aromatic ring substitution or annealing, leading to the systematical increase in the hydrophobic character of the probes. The comparison of the fluorescence behavior of probes 1-3 in a wide variety of solvents with those in phospholipid vesicles revealed that all three probes prefer to reside inside a membrane. Our MD results demonstrate that the probes locate from the glycerol residues and the polar carbonyl groups of phospholipids up to hydrophobic acyl chain units. It has been found that the probe location correlates well with the size of the aromatic moiety, being gradually shifted from 11.1 Å to 7.6 Å from the bilayer center for probes 1 to 3, respectively. Our results may be useful for the design of novel fluorescent probes for fluorescence sensing of specific regions within a lipid membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

PubMed

Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

2010-01-01

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

PubMed

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

A rhodamine chromene-based turn-on fluorescence probe for selectively imaging Cu2+ in living cell

NASA Astrophysics Data System (ADS)

Liu, Wei-Yong; Li, Hai-Ying; Lv, Hong-Shui; Zhao, Bao-Xiang; Miao, Jun-Ying

We describe the development of a rhodamine chromene-based turn-on fluorescence probe to monitor the intracellular Cu2+ level in living cells. The new fluorescent probe with a chlorine group in chromene moiety exhibits good membrane-permeable property than previous reported because the predicted lipophilicity of present probe 4 is stronger than that of methoxyl substituted probe in our previous work (CLogP of 4: 8.313, CLogP of methoxyl substituted probe: 7.706), and a fluorescence response toward Cu2+ under physiological conditions with high sensitivity and selectivity, and facilitates naked-eye detection of Cu2+. The fluorescence intensity was remarkably increased upon the addition of Cu2+ within 1 or 2 min, while the other sixteen metal ions caused no significant effect.

Probing lipid membrane electrostatics

NASA Astrophysics Data System (ADS)

Yang, Yi

The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by <2%. One important application of this technique is to estimate the dipole density of lipid membrane. Electrostatic analysis of DOPC lipid bilayers with the AFM reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipid bilayers. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive manner will be useful in identifying the biological effects of the dipole potential. Finally, heterogeneous model membranes were studied with fluid electric force microscopy (FEFM). Electrostatic mapping was demonstrated with 50 nm resolution. The capabilities of quantitative electrostatic measurement and lateral charge density mapping make AFM a unique and powerful probe of membrane electrostatics.

Fluorescent molecular probes based on excited state prototropism in lipid bilayer membrane

NASA Astrophysics Data System (ADS)

Mohapatra, Monalisa; Mishra, Ashok K.

2012-03-01

Excited state prototropism (ESPT) is observed in molecules having one or more ionizable protons, whose proton transfer efficiency is different in ground and excited states. The interaction of various ESPT molecules like naphthols and intramolecular ESPT (ESIPT) molecules like hydroxyflavones etc. with different microheterogeneous media have been studied in detail and excited state prototropism as a probe concept has been gaining ground. The fluorescence of different prototropic forms of such molecules, on partitioning to an organized medium like lipid bilayer membrane, often show sensitive response to the local environment with respect to the local structure, physical properties and dynamics. Our recent work using 1-naphthol as an ESPT fluorescent molecular probe has shown that the incorporation of monomeric bile salt molecules into lipid bilayer membranes composed from dipalmitoylphosphatidylcholine (DPPC, a lung surfactant) and dimyristoylphosphatidylcholine (DMPC), in solid gel and liquid crystalline phases, induce appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (<= 1 mM) concentrations of the bile salts. The incorporation and location of fisetin, an ESIPT molecule having antioxidant properties, in lipid bilayer membrane has been sensitively monitored from its intrinsic fluorescence behaviour.

Fluorescent probes sensitive to changes in the cholesterol-to-phospholipids molar ratio in human platelet membranes during atherosclerosis

NASA Astrophysics Data System (ADS)

Posokhov, Yevgen

2016-09-01

Environment-sensitive fluorescent probes were used for the spectroscopic visualization of pathological changes in human platelet membranes during cerebral atherosclerosis. It has been estimated that the ratiometric probes 2-(2‧-hydroxyphenyl)-5-phenyl-1,3,4-oxadiazole and 2-phenyl-phenanthr[9,10]oxazole can detect changes in the cholesterol-to-phospholipids molar ratio in human platelet membranes during the disease.

Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

PubMed

Rio, Donald C

2015-03-02

In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity

NASA Astrophysics Data System (ADS)

Chen, Yanan; Qi, Jianguo; Huang, Jing; Zhou, Xiaomin; Niu, Linqiang; Yan, Zhijie; Wang, Jianhong

2018-01-01

Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes.

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential.

PubMed

Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X

2017-03-22

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry-xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe's limitations.

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

PubMed

Papper, V; Medvedeva, N; Fishov, I; Likhtenshtein, G I

2000-01-01

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Nanowire-based single-cell endoscopy

NASA Astrophysics Data System (ADS)

Yan, Ruoxue; Park, Ji-Ho; Choi, Yeonho; Heo, Chul-Joon; Yang, Seung-Man; Lee, Luke P.; Yang, Peidong

2012-03-01

One-dimensional smart probes based on nanowires and nanotubes that can safely penetrate the plasma membrane and enter biological cells are potentially useful in high-resolution and high-throughput gene and drug delivery, biosensing and single-cell electrophysiology. However, using such probes for optical communication across the cellular membrane at the subwavelength level remains limited. Here, we show that a nanowire waveguide attached to the tapered tip of an optical fibre can guide visible light into intracellular compartments of a living mammalian cell, and can also detect optical signals from subcellular regions with high spatial resolution. Furthermore, we show that through light-activated mechanisms the endoscope can deliver payloads into cells with spatial and temporal specificity. Moreover, insertion of the endoscope into cells and illumination of the guided laser did not induce any significant toxicity in the cells.

Optimization of fluorimetric lipid membrane biosensor sensitivity through manipulation of membrane structure and nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine concentration

NASA Astrophysics Data System (ADS)

Shrive, Jason D. A.; Krull, Ulrich J.

1995-01-01

In the work reported here, surface concentrations of 0.027 and 0.073 molecules nm-2 of the fluorescent membrane probe molecule nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) were shown to yield optimum sensitivity for fluorimetric transduction of membrane structural perturbations for lipid membrane-based biosensor development. These optima were obtained through correlation of experimental data with theoretical predictions of optimum surface concentrations based on a model for NBD-PE self quenching previously published by our group. It was also determined that membrane structural heterogeneity improves the sensitivity of NBD-PE labeled membrane transducers. Together with fluorescence microscopy, observations of surface potential change upon compression or expansion of phosphatidylcholine (PC)/phosphatidic acid (PA) monolayers were used to qualitatively indicate the degree of structural heterogeneity in these membranes. It was determined that sub-microscopic domains must exist in microscopically homogeneous egg PC/egg PA membranes in order to facilitate the observed NBD-PE self-quenching responses upon alteration of bulk pH and therefore, membrane surface electrostatics and structure.

DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains☆

PubMed Central

Teague, Heather; Ross, Ron; Harris, Mitchel; Mitchell, Drake C.; Shaikh, Saame Raza

2012-01-01

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo. PMID:22841541

Analyte detection using an active assay

DOEpatents

Morozov, Victor; Bailey, Charles L.; Evanskey, Melissa R.

2010-11-02

Analytes using an active assay may be detected by introducing an analyte solution containing a plurality of analytes to a lacquered membrane. The lacquered membrane may be a membrane having at least one surface treated with a layer of polymers. The lacquered membrane may be semi-permeable to nonanalytes. The layer of polymers may include cross-linked polymers. A plurality of probe molecules may be arrayed and immobilized on the lacquered membrane. An external force may be applied to the analyte solution to move the analytes towards the lacquered membrane. Movement may cause some or all of the analytes to bind to the lacquered membrane. In cases where probe molecules are presented, some or all of the analytes may bind to probe molecules. The direction of the external force may be reversed to remove unbound or weakly bound analytes. Bound analytes may be detected using known detection types.

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

PubMed

Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Egawa, Takahiro; Kobayashi, Chiaki; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Ikegaya, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-10-01

Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential

NASA Astrophysics Data System (ADS)

Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X.

2017-03-01

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry—xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe’s limitations.

A red-emitting indolium fluorescence probe for membranes - flavonoids interactions.

PubMed

Gao, Qingyun; Liu, Han; Ding, Qiongjie; Du, Jinya; Liu, Chunlin; Yang, Wei; Shen, Ping; Yang, Changying

2018-05-01

The red-emitting indolium derivative compound (E)-2-(4-(diphenylamino)styryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (H3) was demonstrated as a sensitive membrane fluorescence probe. The probe located at the interface of liposomes when mixed showed much fluorescence enhancement by inhibiting the twisted intramolecular charge transfer state. After ultrasonic treatment, it penetrated into lipid bilayers with the emissions leveling off and a rather large encapsulation efficiency (71.4%) in liposomes. The ζ-potential and particle size measurement confirmed that the charged indolium group was embedded deeply into lipid bilayers. The probe was then used to monitor the affinities of antioxidant flavonoids for membranes. It was verified that quercetin easily interacted with liposomes and dissociated the probe from the internal lipid within 60 s under the condition of simply mixing. The assessment of binding affinities of six flavonoids and the coincident results with their antioxidation activities indicated that it was a promising membrane probe for the study of drug bio-affinities. Copyright © 2018 John Wiley & Sons, Ltd.

High temperature fiber optic microphone having a pressure-sensing reflective membrane under tensile stress

NASA Technical Reports Server (NTRS)

Zuckerwar, Allan J. (Inventor); Cuomo, Frank W. (Inventor); Robbins, William E. (Inventor); Hopson, Purnell, Jr. (Inventor)

1992-01-01

A fiber optic microphone is provided for measuring fluctuating pressures. An optical fiber probe having at least one transmitting fiber for transmitting light to a pressure-sensing membrane and at least one receiving fiber for receiving light reflected from a stretched membrane is provided. The pressure-sensing membrane may be stretched for high frequency response. Further, a reflecting surface of the pressure-sensing membrane may have dimensions which substantially correspond to dimensions of a cross section of the optical fiber probe. Further, the fiber optic microphone can be made of materials for use in high temperature environments, for example greater than 1000 F. A fiber optic probe is also provided with a backplate for damping membrane motion. The backplate further provides a means for on-line calibration of the microphone.

Fiber optic microphone having a pressure sensing reflective membrane and a voltage source for calibration purpose

NASA Technical Reports Server (NTRS)

Zuckerwar, Allan J. (Inventor); Cuomo, Frank W. (Inventor); Robbins, William E. (Inventor)

1993-01-01

A fiber optic microphone is provided for measuring fluctuating pressures. An optical fiber probe having at least one transmitting fiber for transmitting light to a pressure-sensing membrane and at least one receiving fiber for receiving light reflected from a stretched membrane is provided. The pressure-sensing membrane may be stretched for high frequency response. Further, a reflecting surface of the pressure-sensing membrane may have dimensions which substantially correspond to dimensions of a cross section of the optical fiber probe. Further, the fiber optic microphone can be made of materials for use in high temperature environments, for example greater than 1000 F. A fiber optic probe is also provided with a back plate for damping membrane motion. The back plate further provides a means for on-line calibration of the microphone.

Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

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

Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining wasmore » determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.« less

A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

NASA Astrophysics Data System (ADS)

AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

2015-01-01

Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06102k

Intraocular laser surgical probe for membrane disruption by laser-induced breakdown.

PubMed

Hammer, D X; Noojin, G D; Thomas, R J; Clary, C E; Rockwell, B A; Toth, C A; Roach, W P

1997-03-01

A fiber probe has been designed as a surgical aid to cut intraocular membranes with laser-induced breakdown as the mechanism. The design of the intraocular laser surgical probe is discussed. A preliminary retinal damage distance has been calculated with breakdown threshold, spot size, and shielding measurements. Collateral mechanical-damage effects caused by shock wave and cavitation are discussed.

Far Western: probing membranes.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

Core-based lipid nanoparticles as a nanoplatform for delivery of near-infrared fluorescent imaging agents.

PubMed

Anikeeva, Nadia; Sykulev, Yuri; Delikatny, Edward J; Popov, Anatoliy V

2014-01-01

Pyropheophorbide a (Pyro) is a near-infrared (NIR) fluorescent dye and photosensitizer with high quantum yield that makes the dye suitable for tumor treatment both as an imaging and therapy agent. We have designed and synthesized a series of a Pyro-based NIR probes, based on the conjugation of Pyro with lipids. The nature of our probes requires the use of a lipophilic carrier to deliver the probes to cancer cell membranes. To address this, we have utilized lipid-based nanoparticles (LNPs) consisting of PEGylated lipids, which form the nanoparticle shell, and a lipid core. To endow the LNPs with targeting properties, nitrilotriacetic acid (NTA) lipids were included in the composition that enables the non-covalent attachment of His-tag targeting proteins preserving their functional activity. We found that the nature of the core molecules influence the nanoparticle size, shelf-life and stability at physiological temperature. Two different Pyro-lipid conjugates were loaded either into the core or shell of the LNPs. The conjugates revealed differential ability to be accumulated in the cell membrane of the target cells with time. Thus, the modular organization of the core-shell LNPs allows facile adjustment of their composition with goal to fine tuning the nanoparticle properties for in vivo application.

Core-based lipid nanoparticles as a nanoplatform for delivery of near-infrared fluorescent imaging agents

PubMed Central

Anikeeva, Nadia; Sykulev, Yuri; Delikatny, Edward J; Popov, Anatoliy V

2014-01-01

Pyropheophorbide a (Pyro) is a near-infrared (NIR) fluorescent dye and photosensitizer with high quantum yield that makes the dye suitable for tumor treatment both as an imaging and therapy agent. We have designed and synthesized a series of a Pyro-based NIR probes, based on the conjugation of Pyro with lipids. The nature of our probes requires the use of a lipophilic carrier to deliver the probes to cancer cell membranes. To address this, we have utilized lipid-based nanoparticles (LNPs) consisting of PEGylated lipids, which form the nanoparticle shell, and a lipid core. To endow the LNPs with targeting properties, nitrilotriacetic acid (NTA) lipids were included in the composition that enables the non-covalent attachment of His-tag targeting proteins preserving their functional activity. We found that the nature of the core molecules influence the nanoparticle size, shelf-life and stability at physiological temperature. Two different Pyro-lipid conjugates were loaded either into the core or shell of the LNPs. The conjugates revealed differential ability to be accumulated in the cell membrane of the target cells with time. Thus, the modular organization of the core-shell LNPs allows facile adjustment of their composition with goal to fine tuning the nanoparticle properties for in vivo application. PMID:25250201

Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP.

PubMed

Lundby, Alicia; Akemann, Walther; Knöpfel, Thomas

2010-11-01

A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.

Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches

PubMed Central

Malacrida, Leonel; Gratton, Enrico; Jameson, David M

2016-01-01

In this note, we present a discussion of the advantages and scope of model-free analysis methods applied to the popular solvatochromic probe LAURDAN, which is widely used as an environmental probe to study dynamics and structure in membranes. In particular, we compare and contrast the generalized polarization approach with the spectral phasor approach. To illustrate our points we utilize several model membrane systems containing pure lipid phases and, in some cases, cholesterol or surfactants. We demonstrate that the spectral phasor method offers definitive advantages in the case of complex systems. PMID:27182438

Gas-Permeable Membrane-Based Conductivity Probe Capable of In Situ Real-Time Monitoring of Ammonia in Aquatic Environments.

PubMed

Li, Tianling; Panther, Jared; Qiu, Yuan; Liu, Chang; Huang, Jianyin; Wu, Yonghong; Wong, Po Keung; An, Taicheng; Zhang, Shanqing; Zhao, Huijun

2017-11-21

Aquatic ammonia has toxic effects on aquatic life. This work reports a gas-permeable membrane-based conductivity probe (GPMCP) developed for real-time monitoring of ammonia in aquatic environments. The GPMCP innovatively combines a gas-permeable membrane with a boric acid receiving phase to selectively extract ammonia from samples and form ammonium at the inner membrane interface. The rate of the receiving phase conductivity increase is directly proportional to the instantaneous ammonia concentration in the sample, which can be rapidly and sensitively determined by the embedded conductivity detector. A precalibration strategy was developed to eliminate the need for an ongoing calibration. The analytical principle and GPMCP performance were systematically validated. The laboratory results showed that ammonia concentrations ranging from 2 to 50 000 μg L -1 can be detected. The field deployment results demonstrated the GPMCP's ability to obtain high-resolution continuous ammonia concentration profiles and the absolute average ammonia concentration over a prolonged deployment period. By inputting the temperature and pH data, the ammonium concentration can be simultaneously derived from the corresponding ammonia concentration. The GPMCP embeds a sophisticated analytical principle with the inherent advantages of high selectivity, sensitivity, and accuracy, and it can be used as an effective tool for long-term, large-scale, aquatic-environment assessments.

Membrane-based electrochemical nanobiosensor for Escherichia coli detection and analysis of cells viability.

PubMed

Cheng, Ming Soon; Lau, Suk Hiang; Chow, Vincent T; Toh, Chee-Seng

2011-08-01

A sensitive and selective membrane-based electrochemical nanobiosensor is developed for specific quantitative label-free detection of Escherichia coli (E. coli) cells and analysis of viable but nonculturable (VBNC) E. coli cells which remain mostly undetected using current methods. The sensing mechanism relies on the blocking of nanochannels of a nanoporous alumina-membrane modified electrode, upon the formation of immune complexes at the nanoporous membrane. The resulting obstacle to diffusive mass transfer of a redox probe in the analysis solution to the underlying platinum electrode reduces the Faradaic signal response of the biosensor, measured using cyclic voltammetry. Antibody loading under conditions of varying antibody concentrations and pHs are optimized. The biosensor gives a low detection limit of 22 cfu mL(-1) (R(2) = 0.999) over a wide linear working range of 10 to 10(6) cfu mL(-1). It is specific toward E. coli with minimal cross-reactivity to two other pathogenic bacteria (commonly found in waters). Relative standard deviation (RSD) for triplicate measurements of 2.5% indicates reasonably useful level of reproducibility. Differentiation of live, VBNC, and dead cells are carried out after the cell capture and quantitation step, by simple monitoring of the cells' enzyme activity using the same redox probe in the analysis solution, in the presence of glucose.

Using membrane composition to fine-tune the pKa of an optical liposome pH sensor.

PubMed

Clear, Kasey J; Virga, Katelyn; Gray, Lawrence; Smith, Bradley D

2016-04-14

Liposomes containing membrane-anchored pH-sensitive optical probes are valuable sensors for monitoring pH in various biomedical samples. The dynamic range of the sensor is maximized when the probe p K a is close to the expected sample pH. While some biomedical samples are close to neutral pH there are several circumstances where the pH is 1 or 2 units lower. Thus, there is a need to fine-tune the probe p K a in a predictable way. This investigation examined two lipid-conjugated optical probes, each with appended deep-red cyanine dyes containing indoline nitrogen atoms that are protonated in acid. The presence of anionic phospholipids in the liposomes stabilized the protonated probes and increased the probe p K a values by < 1 unit. The results show that rational modification of the membrane composition is a general non-covalent way to fine-tune the p K a of an optical liposome sensor for optimal pH sensing performance.

The fluorescence lifetime of BRI1-GFP as probe for the noninvasive determination of the membrane potential in living cells

NASA Astrophysics Data System (ADS)

Elgass, K.; Caesar, K.; Schleifenbaum, F.; Meixner, A. J.; Harter, K.

2010-02-01

As the excited state lifetime of a fluorescent molecule depends on its environment, it is possible to use it as a probe for physico-chemical parameters of the surrounding medium. Whereas this is well known for many solid guest/host systems, only few reports of quantitative, temporal resolved in vivo studies to monitor the nano-environment for a protein-coupled chromophore such as GFP are known from literature. Here we present a novel approach to determine the membrane potential of living (plant) cells based on the fluorescence lifetime (FLT) analysis of membrane-located GFP. By using confocal sample scanning microscopy (CSSM) combined with fluorescence lifetime imaging microscopy, we recently showed that the phytohormone brassinolide (BL) induces cell wall expansion and a decrease in the FLT of the BRI1-GFP in living cells of Arabidopsis thaliana seedlings. BRI1 is the dominant functional receptor for BL in Arabidopsis and locates to the plasma membrane. Although the dependence of the FLT of GFP on its physico-chemical environment such as pH-value, refractive index and pressure has been reported, the observed FLT decrease of BRI1-GFP in response to BL application could not be explained by these parameters. However, our in vivo FLT and CSSM analyses indicate that the BLinduced change in the FLT of BRI1-GFP is caused by hyperpolarisation of the plasma membrane (Em). Thus, our results indicate that BRI1-GFP serves as sensitive and non-invasive probe for recording the Em of the plasma membrane in living plant cells with high spatio-temporal resolution.

Monitoring probe for groundwater flow

DOEpatents

Looney, Brian B.; Ballard, Sanford

1994-01-01

A monitoring probe for detecting groundwater migration. The monitor features a cylinder made of a permeable membrane carrying an array of electrical conductivity sensors on its outer surface. The cylinder is filled with a fluid that has a conductivity different than the groundwater. The probe is placed in the ground at an area of interest to be monitored. The fluid, typically saltwater, diffuses through the permeable membrane into the groundwater. The flow of groundwater passing around the permeable membrane walls of the cylinder carries the conductive fluid in the same general direction and distorts the conductivity field measured by the sensors. The degree of distortion from top to bottom and around the probe is precisely related to the vertical and horizontal flow rates, respectively. The electrical conductivities measured by the sensors about the outer surface of the probe are analyzed to determine the rate and direction of the groundwater flow.

Monitoring probe for groundwater flow

DOEpatents

Looney, B.B.; Ballard, S.

1994-08-23

A monitoring probe for detecting groundwater migration is disclosed. The monitor features a cylinder made of a permeable membrane carrying an array of electrical conductivity sensors on its outer surface. The cylinder is filled with a fluid that has a conductivity different than the groundwater. The probe is placed in the ground at an area of interest to be monitored. The fluid, typically saltwater, diffuses through the permeable membrane into the groundwater. The flow of groundwater passing around the permeable membrane walls of the cylinder carries the conductive fluid in the same general direction and distorts the conductivity field measured by the sensors. The degree of distortion from top to bottom and around the probe is precisely related to the vertical and horizontal flow rates, respectively. The electrical conductivities measured by the sensors about the outer surface of the probe are analyzed to determine the rate and direction of the groundwater flow. 4 figs.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

PubMed Central

Zhang, Ming; Chakraborty, Subhasish K.; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

2015-01-01

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene. PMID:26348895

Electrospin-coating of nitrocellulose membrane enhances sensitivity in nucleic acid-based lateral flow assay.

PubMed

Yew, Chee-Hong Takahiro; Azari, Pedram; Choi, Jane Ru; Li, Fei; Pingguan-Murphy, Belinda

2018-06-07

Point-of-care biosensors are important tools developed to aid medical diagnosis and testing, food safety and environmental monitoring. Paper-based biosensors, especially nucleic acid-based lateral flow assays (LFA), are affordable, simple to produce and easy to use in remote settings. However, the sensitivity of such assays to infectious diseases has always been a restrictive challenge. Here, we have successfully electrospun polycaprolactone (PCL) on nitrocellulose (NC) membrane to form a hydrophobic coating to reduce the flow rate and increase the interaction rate between the targets and gold nanoparticles-detecting probes conjugates, resulting in the binding of more complexes to the capture probes. With this approach, the sensitivity of the PCL electrospin-coated test strip has been increased by approximately ten-fold as compared to the unmodified test strip. As a proof of concept, this approach holds great potential for sensitive detection of targets at point-of-care testing. Copyright © 2018 Elsevier B.V. All rights reserved.

Measurement Techniques for Flow Diagnostic in ITAM Impulse Wind Tunnels

DTIC Science & Technology

2010-04-01

time of wind - tunnel operation, so that oscillations caused by initial shock loads could decay and a comparatively long time period with constant flow...Flow Diagnostic in ITAM Impulse Wind Tunnels 7 - 4 RTO-EN-AVT-186 A strain-gauge pressure probe is an elastic element (membrane) in a sealed...Diagnostic in ITAM Impulse Wind Tunnels RTO-EN-AVT-186 7 - 5 probes are individually calibrated. Piezoelectric pressure gauges are based

Deuterated fatty acids as Raman spectroscopic probes of membrane structure.

PubMed

Mendelsohn, R; Sunder, S; Bernstein, H J

1976-09-07

Raman spectra are reported for the C-D stretching region of stearic acid-d35 bound in egg lecithin multilayers. The temperature dependence of the spectra shows that the linewidth of the C-D stretching bands is a sensitive and non-perturbative probe of membrane hydrocarbon chain conformation. The utility of this approach for studying lipid conformation in membranes containing a significant fraction of non-lipid component is discussed.

Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

PubMed Central

Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

2013-01-01

Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

Base Stability of Aminocyclopropeniums

DTIC Science & Technology

2017-11-01

stability, a series of aminocyclopropeniums were synthesized and their base stability probed in situ using time -resolved proton nuclear magnetic resonance...reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions...tested for their utility in anion exchange membranes for alkaline fuel cells. A series of aminocyclopropeniums were synthesized and their base

Changes in lipid membranes may trigger amyloid toxicity in Alzheimer's disease

PubMed Central

Drolle, Elizabeth; Negoda, Alexander; Hammond, Keely; Pavlov, Evgeny

2017-01-01

Amyloid-beta peptides (Aβ), implicated in Alzheimer’s disease (AD), interact with the cellular membrane and induce amyloid toxicity. The composition of cellular membranes changes in aging and AD. We designed multi-component lipid models to mimic healthy and diseased states of the neuronal membrane. Using atomic force microscopy (AFM), Kelvin probe force microscopy (KPFM) and black lipid membrane (BLM) techniques, we demonstrated that these model membranes differ in their nanoscale structure and physical properties, and interact differently with Aβ1–42. Based on our data, we propose a new hypothesis that changes in lipid membrane due to aging and AD may trigger amyloid toxicity through electrostatic mechanisms, similar to the accepted mechanism of antimicrobial peptide action. Understanding the role of the membrane changes as a key activating amyloid toxicity may aid in the development of a new avenue for the prevention and treatment of AD. PMID:28767712

Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes.

PubMed

Niko, Yosuke; Didier, Pascal; Mely, Yves; Konishi, Gen-ichi; Klymchenko, Andrey S

2016-01-11

Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

[Utilization of nylon membranes for specific isolation and characterization of verotoxin-producing Escherichia coli using DNA probes].

PubMed

Gallien, P; Klie, H; Perlberg, K W; Protz, D

1996-01-01

A method for specific isolation of VT(+)-strains in raw milk is given. DNA-hybridization technique with DIG-labeled PCR-amplificates as probes are the basis. No background is seen by using "DIG Easy Hyb" solution and nylon membranes for colony- and plaque-hybridization (Boehringer Mannheim GmbH). Marked colonies are visible on the membranes after detection. So it is possible to select these colonies from a masterplate. The results are available within one day (without enrichment and membrane preparation). After stripping the membranes can be used for a new hybridisation to detect another factor of virulence.

Specific DNA duplex formation at an artificial lipid bilayer: towards a new DNA biosensor technology.

PubMed

Werz, Emma; Korneev, Sergei; Montilla-Martinez, Malayko; Wagner, Richard; Hemmler, Roland; Walter, Claudius; Eisfeld, Jörg; Gall, Karsten; Rosemeyer, Helmut

2012-02-01

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H(2) O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5'-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer. Injection of a cyanine-5-labeled target DNA sequence, which is complementary to only one of the oligonucleotide probes, into the cis-channel, followed by a thorough perfusion, leads to an immobilization of the labeled complementary oligonucleotide on the membrane as detected by single-molecule fluorescence spectroscopy and microscopy. In the case of fluorescent but non-complementary DNA sequences, no immobilized fluorescent oligonucleotide duplex could be detected on the membrane. This clearly verifies a specific duplex formation at the membrane interface. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

A new fluorescent pH probe for imaging lysosomes in living cells.

PubMed

Lv, Hong-Shui; Huang, Shu-Ya; Xu, Yu; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

2014-01-15

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure-activity relationships of succinimidyl-Cys-C(O)-Glu derivatives with different near-infrared fluorophores as optical imaging probes for prostate-specific membrane antigen.

PubMed

Matsuoka, Daiko; Watanabe, Hiroyuki; Shimizu, Yoichi; Kimura, Hiroyuki; Yagi, Yusuke; Kawai, Ryoko; Ono, Masahiro; Saji, Hideo

2018-05-15

Prostate-specific membrane antigen (PSMA), which is overexpressed in malignant prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported a PSMA imaging probe, 800CW-SCE, based on succinimidyl-Cys-C(O)-Glu (SCE) for optical imaging of PCa. In this study, we investigated the structure-activity relationships of novel SCE derivatives with five different near-infrared (NIR) fluorophores (IRDye 680LT, IRDye 750, Indocyanine Green, Cyanine 5.5, and Cyanine 7) as optical imaging probes targeting PSMA. An in vitro binding assay revealed that 800CW-SCE, 680LT-SCE, and 750-SCE exhibited higher binding affinity than 2-PMPA, which is known as a PSMA inhibitor. These three SCE derivatives were internalized into PSMA-positive cells (LNCaP cells) but not into PSMA-negative cells (PC-3 cells). In the in vivo imaging study, 800CW-SCE and 750-SCE were highly accumulated in LNCaP tumors but not in PC-3 tumors, and the ratio of LNCaP/PC-3 accumulation of 800CW-SCE was higher than that of 750-SCE. The present study may provide valuable molecular design information for the future development of new PSMA imaging probes based on the SCE scaffold. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes*

PubMed Central

Zhou, Yong; Plowman, Sarah J.; Lichtenberger, Lenard M.; Hancock, John F.

2010-01-01

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase seperation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane. PMID:20826816

Membrane order in the plasma membrane and endocytic recycling compartment.

PubMed

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Membrane order in the plasma membrane and endocytic recycling compartment

PubMed Central

Iaea, David B.; Maxfield, Frederick R.

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles. PMID:29125865

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

PubMed Central

Senning, Eric N.; Aman, Teresa K.

2016-01-01

Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane. PMID:26755772

Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

2011-03-01

Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

Super-resolved FT-IR spectroscopy: Strategies, challenges, and opportunities for membrane biophysics.

PubMed

Li, Jessica J; Yip, Christopher M

2013-10-01

Direct correlation of molecular conformation with local structure is critical to studies of protein- and peptide-membrane interactions, particularly in the context of membrane-facilitated aggregation, and disruption or disordering. Infrared spectroscopy has long been a mainstay for determining molecular conformation, following folding dynamics, and characterizing reactions. While tremendous advances have been made in improving the spectral and temporal resolution of infrared spectroscopy, it has only been with the introduction of scanned-probe techniques that exploit the raster-scanning tip as either a source, scattering tool, or measurement probe that researchers have been able to obtain sub-diffraction limit IR spectra. This review will examine the history of correlated scanned-probe IR spectroscopies, from their inception to their use in studies of molecular aggregates, membrane domains, and cellular structures. The challenges and opportunities that these platforms present for examining dynamic phenomena will be discussed. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications.

PubMed

Jiang, Xiqian; Wang, Lingfei; Carroll, Shaina L; Chen, Jianwei; Wang, Meng C; Wang, Jin

2018-02-16

The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 00, 000-000.

Nanopipette Delivery of Individual Molecules to Cellular Compartments for Single-Molecule Fluorescence Tracking

PubMed Central

Bruckbauer, Andreas; James, Peter; Zhou, Dejian; Yoon, Ji Won; Excell, David; Korchev, Yuri; Jones, Roy; Klenerman, David

2007-01-01

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane. PMID:17631532

Effect of a glucose-triazole-hydrogenated cardanol conjugate on lipid bilayer membrane organization and thermotropic phase transition

NASA Astrophysics Data System (ADS)

Swain, Jitendriya; Kamalraj, M.; Surya Prakash Rao, H.; Mishra, Ashok K.

2015-02-01

This work focuses on the membrane perturbation, solubilisation and thermotropic phase transition process of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) MLVs induced by a glucose-triazole-hydrogenated cardanol conjugate (GTHCC). GTHCC is a recently introduced non toxic sugar derivative. Differential scanning calorimetry (DSC) and fluorescence molecular probe based techniques have been used to understand the concentration dependent membrane perturbation, solubilisation and thermotropic phase transition process of DPPC MLVs. The phase transition temperature of DPPC MLVs decreases with increase in mol% of GTHCC. At higher concentration above 10 mol%, GTHCC was significantly perturbed the membrane organization. The intrinsic fluorescence of GTHCC is also found to be sensitive towards phase behaviour and changes in membrane organization of DPPC MLVs.

Listening to membrane potential: photoacoustic voltage-sensitive dye recording.

PubMed

Zhang, Haichong K; Yan, Ping; Kang, Jeeun; Abou, Diane S; Le, Hanh N D; Jha, Abhinav K; Thorek, Daniel L J; Kang, Jin U; Rahmim, Arman; Wong, Dean F; Boctor, Emad M; Loew, Leslie M

2017-04-01

Voltage-sensitive dyes (VSDs) are designed to monitor membrane potential by detecting fluorescence changes in response to neuronal or muscle electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. By contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near-infrared light excitation and ultrasound detection. Here, we show that voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. We synthesized a near-infrared photoacoustic VSD (PA-VSD), whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. A theoretical model accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate PA voltage sensing but also emphasize the interplay of both fluorescence and absorbance properties in the design of optimized PA probes. Together, our results demonstrate PA sensing as a potential new modality for recording and external imaging of electrophysiological and neurochemical events in the brain.

Listening to membrane potential: photoacoustic voltage-sensitive dye recording

NASA Astrophysics Data System (ADS)

Zhang, Haichong K.; Yan, Ping; Kang, Jeeun; Abou, Diane S.; Le, Hanh N. D.; Jha, Abhinav K.; Thorek, Daniel L. J.; Kang, Jin U.; Rahmim, Arman; Wong, Dean F.; Boctor, Emad M.; Loew, Leslie M.

2017-04-01

Voltage-sensitive dyes (VSDs) are designed to monitor membrane potential by detecting fluorescence changes in response to neuronal or muscle electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. By contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near-infrared light excitation and ultrasound detection. Here, we show that voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. We synthesized a near-infrared photoacoustic VSD (PA-VSD), whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. A theoretical model accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate PA voltage sensing but also emphasize the interplay of both fluorescence and absorbance properties in the design of optimized PA probes. Together, our results demonstrate PA sensing as a potential new modality for recording and external imaging of electrophysiological and neurochemical events in the brain.

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

PubMed Central

Moen, Erick K.; Ibey, Bennett L.; Beier, Hope T.

2014-01-01

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. PMID:24853757

Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

PubMed

Moen, Erick K; Ibey, Bennett L; Beier, Hope T

2014-05-20

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

SYTO probes: markers of apoptotic cell demise.

PubMed

Wlodkowic, Donald; Skommer, Joanna

2007-10-01

As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high-throughput analytical tools to detect diverse cell demise modes. Patented DNA-binding SYTO probes, for example, are gaining increasing interest as easy-to-use markers of caspase-dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7-aminoactinomycin D (7-AAD).

Optical Antenna-Based Fluorescence Correlation Spectroscopy to Probe the Nanoscale Dynamics of Biological Membranes.

PubMed

Winkler, Pamina M; Regmi, Raju; Flauraud, Valentin; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F

2018-01-04

The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This nonrandom organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nanoassemblies enriched by cholesterol, sphingolipids, and certain types of proteins. Investigating these nanoassemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of common microscopes. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit of light. In this Perspective, we discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to monitor microsecond dynamics at nanoscale spatial dimensions. These new developments offer numerous opportunities to investigate lipid and protein dynamics in both mimetic and native biological membranes.

Development of a Methodology for the Characterisation of Air-coupled Ultrasound Probes

NASA Astrophysics Data System (ADS)

Pietroni, Paolo; Marco Revel, Gian

2010-05-01

This study is aimed at developing a technique for the characterisation of air-coupled ultrasound probes, starting from the analysis of the mechanical behaviour of the probe membrane. The vibratory behaviour of the emission membrane is studied using laser-Doppler vibrometry techniques with high frequency demodulation system (20 MHz). The determination of the vibration provides information which are useful for the assessment of the performance of the probe, in particular concerning the Quality factor and the portion of the membrane which really contributes to the emission. During the second step the results of the vibration measurements are used to calculate, by means of numerical boundary element method, the ultrasound beam emitted in terms of intensity in space. The obtained field is compared with the direct measurements carried out by scanning with the receiver probe and a pinhole plate. This comparison allows the potential and the problems of the two different characterisation techniques to be determined, even if the pinhole technique (which is currently considered the state of the art) cannot be used as an absolute reference. This study appears to be useful for paving the way for a new methodology for the calibration of air-coupled ultrasound probes, which potentially could be used not only to improve the probe manufacturing process, but also to control conformity to specifications.

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

NASA Astrophysics Data System (ADS)

Jia, Lihui; Liang, Shuang; Sackett, Kelly; Xie, Li; Ghosh, Ujjayini; Weliky, David P.

2015-04-01

Rotational-echo double-resonance (REDOR) solid-state NMR is applied to probe the membrane locations of specific residues of membrane proteins. Couplings are measured between protein 13CO nuclei and membrane lipid or cholesterol 2H and 31P nuclei. Specific 13CO labeling is used to enable unambiguous assignment and 2H labeling covers a small region of the lipid or cholesterol molecule. The 13CO-31P and 13CO-2H REDOR respectively probe proximity to the membrane headgroup region and proximity to specific insertion depths within the membrane hydrocarbon core. One strength of the REDOR approach is use of chemically-native proteins and membrane components. The conventional REDOR pulse sequence with 100 kHz 2H π pulses is robust with respect to the 2H quadrupolar anisotropy. The 2H T1's are comparable to the longer dephasing times (τ's) and this leads to exponential rather than sigmoidal REDOR buildups. The 13CO-2H buildups are well-fitted to A × (1 - e-γτ) where A and γ are fitting parameters that are correlated as the fraction of molecules (A) with effective 13CO-2H coupling d = 3γ/2. The REDOR approach is applied to probe the membrane locations of the "fusion peptide" regions of the HIV gp41 and influenza virus hemagglutinin proteins which both catalyze joining of the viral and host cell membranes during initial infection of the cell. The HIV fusion peptide forms an intermolecular antiparallel β sheet and the REDOR data support major deeply-inserted and minor shallowly-inserted molecular populations. A significant fraction of the influenza fusion peptide molecules form a tight hairpin with antiparallel N- and C-α helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the β and α fusion peptide structures may be relevant for fusion catalysis via the resultant local perturbation of the membrane bilayer. Future applications of the REDOR approach may include samples that contain cell membrane extracts and use of lower temperatures and dynamic nuclear polarization to reduce data acquisition times.

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes.

PubMed

Rakers, Lena; Grill, David; Matos, Anna L L; Wulff, Stephanie; Wang, Da; Börgel, Jonas; Körsgen, Martin; Arlinghaus, Heinrich F; Galla, Hans-Joachim; Gerke, Volker; Glorius, Frank

2018-05-01

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Artificial Lipid Membranes: Past, Present, and Future

PubMed Central

Siontorou, Christina G.; Nikoleli, Georgia-Paraskevi; Nikolelis, Dimitrios P.

2017-01-01

The multifaceted role of biological membranes prompted early the development of artificial lipid-based models with a primary view of reconstituting the natural functions in vitro so as to study and exploit chemoreception for sensor engineering. Over the years, a fair amount of knowledge on the artificial lipid membranes, as both, suspended or supported lipid films and liposomes, has been disseminated and has helped to diversify and expand initial scopes. Artificial lipid membranes can be constructed by several methods, stabilized by various means, functionalized in a variety of ways, experimented upon intensively, and broadly utilized in sensor development, drug testing, drug discovery or as molecular tools and research probes for elucidating the mechanics and the mechanisms of biological membranes. This paper reviews the state-of-the-art, discusses the diversity of applications, and presents future perspectives. The newly-introduced field of artificial cells further broadens the applicability of artificial membranes in studying the evolution of life. PMID:28933723

On scattered waves and lipid domains: detecting membrane rafts with X-rays and neutrons

DOE PAGES

Marquardt, Drew; Heberle, Frederick A.; Nickels, Jonathan D.; ...

2015-09-21

In order to understand the biological role of lipids in cell membranes, it is necessary to determine the mesoscopic structure of well-defined model membrane systems. Neutron and X-ray scattering are non-invasive, probe-free techniques that have been used extensively in such systems to probe length scales ranging from angstroms to microns, and dynamics occurring over picosecond to millisecond time scales. Finally, recent developments in the area of phase separated lipid systems mimicking membrane rafts will be presented, and the underlying concepts of the different scattering techniques used to study them will be discussed in detail.

Probing Membrane Order and Topography in Supported Lipid Bilayers by Combined Polarized Total Internal Reflection Fluorescence-Atomic Force Microscopy

PubMed Central

Oreopoulos, John; Yip, Christopher M.

2009-01-01

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C20 or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions. PMID:19254557

Probing the interaction between nanoparticles and lipid membranes by quartz crystal microbalance with dissipation monitoring

NASA Astrophysics Data System (ADS)

Yousefi, Nariman; Tufenkji, Nathalie

2016-12-01

There is increasing interest in using quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the interaction of nanoparticles (NPs) with model surfaces. The high sensitivity, ease of use and the ability to monitor interactions in real-time has made it a popular technique for colloid chemists, biologists, bioengineers and biophysicists. QCM-D has been recently used to probe the interaction of NPs with supported lipid bilayers (SLBs) as model cell membranes. The interaction of NPs with SLBs is highly influenced by the quality of the lipid bilayers. Unlike many surface sensitive techniques, using QCM-D, the quality of SLBs can be assessed in real-time, hence QCM-D studies on SLB-NP interactions are less prone to the artefacts arising from bilayers that are not well formed. The ease of use and commercial availability of a wide range of sensor surfaces also have made QCM-D a versatile tool for studying NP interactions with lipid bilayers. In this review, we summarize the state-of-the-art on QCM-D based techniques for probing the interactions of NPs with lipid bilayers.

Probing microscopic material properties inside simulated membranes through spatially resolved three-dimensional local pressure fields and surface tensions

PubMed Central

Kasson, Peter M.; Hess, Berk; Lindahl, Erik

2013-01-01

Cellular lipid membranes are spatially inhomogeneous soft materials. Materials properties such as pressure and surface tension thus show important microscopic-scale variation that is critical to many biological functions. We present a means to calculate pressure and surface tension in a 3D-resolved manner within molecular-dynamics simulations and show how such measurements can yield important insight. We also present the first corrections to local virial and pressure fields to account for the constraints typically used in lipid simulations that otherwise cause problems in highly oriented systems such as bilayers. Based on simulations of an asymmetric bacterial ion channel in a POPC bilayer, we demonstrate how 3D-resolved pressure can probe for both short-range and long-range effects from the protein on the membrane environment. We also show how surface tension is a sensitive metric for inter-leaflet equilibrium and can be used to detect even subtle imbalances between bilayer leaflets in a membrane-protein simulation. Since surface tension is known to modulate the function of many proteins, this effect is an important consideration for predictions of ion channel function. We outline a strategy by which our local pressure measurements, which we make available within a version of the GROMACS simulation package, may be used to design optimally equilibrated membrane-protein simulations. PMID:23318532

Nanofabrication technique based on localized photocatalytic reactions using a TiO2-coated atomic force microscopy probe

NASA Astrophysics Data System (ADS)

Shibata, Takayuki; Iio, Naohiro; Furukawa, Hiromi; Nagai, Moeto

2017-02-01

We performed a fundamental study on the photocatalytic degradation of fluorescently labeled DNA molecules immobilized on titanium dioxide (TiO2) thin films under ultraviolet irradiation. The films were prepared by the electrochemical anodization of Ti thin films sputtered on silicon substrates. We also confirmed that the photocurrent arising from the photocatalytic oxidation of DNA molecules can be detected during this process. We then demonstrated an atomic force microscopy (AFM)-based nanofabrication technique by employing TiO2-coated AFM probes to penetrate living cell membranes under near-physiological conditions for minimally invasive intracellular delivery.

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order.

PubMed

Aron, Miles; Browning, Richard; Carugo, Dario; Sezgin, Erdinc; Bernardino de la Serna, Jorge; Eggeling, Christian; Stride, Eleanor

2017-05-12

Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes. Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox .

Highly water-soluble BODIPY-based fluorescent probe for sensitive and selective detection of nitric oxide in living cells.

PubMed

Vegesna, Giri K; Sripathi, Srinivas R; Zhang, Jingtuo; Zhu, Shilei; He, Weilue; Luo, Fen-Tair; Jahng, Wan Jin; Frost, Megan; Liu, Haiying

2013-05-22

A highly water-soluble BODIPY dye bearing electron-rich o-diaminophenyl groups at 2,6-positions was prepared as a highly sensitive and selective fluorescent probe for detection of nitric oxide (NO) in living cells. The fluorescent probe displays an extremely weak fluorescence with fluorescence quantum yield of 0.001 in 10 mM phosphate buffer (pH 7.0) in the absence of NO as two electron-rich o-diaminophenyl groups at 2,6-positions significantly quench the fluorescence of the BODIPY dye via photoinduced electron transfer mechanism. The presence of NO in cells enhances the dye fluorescence dramatically. The fluorescent probe demonstrates excellent water solubility, membrane permeability, and compatibility with living cells for sensitive detection of NO.

Long-term Calibration Considerations during Subcutaneous Microdialysis Sampling in Mobile Rats

PubMed Central

Mou, Xiaodun; Lennartz, Michelle; Loegering, Daniel J.; Stenken, Julie A.

2010-01-01

The level at which implanted sensors and sampling devices maintain their calibration is an important research area. In this work, microdialysis probes with identical geometry and different membranes, polycarbonate/polyether (PC) or polyethersulfone (PES), were used with internal standards (vitamin B12 (MW 1355), antipyrine (MW 188) and 2-deoxyglucose (2-DG, MW 164)) and endogenous glucose to investigate changes in their long-term calibration after implantation into the subcutaneous space of Sprague-Dawley rats. Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for antipyrine and 2-DG was not altered throughout the implant lifetime for either PC- or PES-membranes. Yet, Vitamin B12 extraction efficiency and collected glucose concentrations decreased during the implant lifetime. Antipyrine was administered i.v. and its concentrations obtained in both PC-and PES-membrane probes were significantly reduced between the implant day and seven (PC) or 10 (PES) days post implantation suggesting that solute supply is critical for in vivo extraction efficiency. For the low molecular weight solutes such as antipyrine and glucose, localized delivery is not affected by the foreign body reaction, but recovery is significantly reduced. For Vitamin B12, a larger solute, the fibrotic capsule formed around the probe significantly restricts diffusion from the implanted microdialysis probes. PMID:20223515

Docosahexaenoic acid conjugated near infrared flourescence probe for in vivo early tumor diagnosis

NASA Astrophysics Data System (ADS)

Li, Siwen; Cao, Jie; Qin, Jingyi; Zhang, Xin; Achilefu, Samuel; Qian, Zhiyu; Gu, Yueqing

2013-02-01

Docosahexaenoic acid(DHA) is an omega-3 C22 natural fatty acid with six cis double bonds and as a constituent of membranes used as a precursor for metabolic and biochemical path ways. In this manuscript,we describe the synthesis of near-infrared(NIR) flourescence ICG-Der-01 labeled DHA for in vitro and vivo tumor targeting.The structure of the probe was intensively characterized by UV and MS. The in vitro and vivo tumor targeting abilities of the DHA-based NIR probes were investigeted in MCF-7 cells and MCF-7 xenograft mice model differently by confocal microscopy and CCD camera. The cell cytotoxicity were tested in tumor cells MCF-7 .The results shows that the DHA-based NIR probes have high affinity with the tumor both in vitro and vivo.In addition ,we also found that the DHA-based NIR probes have the apparent cytotoxicity on MCF-7 cells .which demonstrated that DHA was conjugated with other antitumor drug could increase the abilities of antirumor efficacy .So DHA-ICG-Der-01 is a promising optical agent for diagnosis of tumors especially in their early stage.

An alternative method for processing northern blots after capillary transfer.

PubMed

Nilsen, Timothy W

2015-03-02

Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane. © 2015 Cold Spring Harbor Laboratory Press.

Transport behavior of hairless mouse skin during constant current DC iontophoresis, part 2: iontophoresis of nonionic molecules with cotransport of polystyrene sulfonate oligomers.

PubMed

Liddell, Mark R; Li, S Kevin; Higuchi, William I

2011-07-01

The purpose of this study was to characterize changes that occur in the iontophoretic transport of nonionic probe permeants in hairless mouse skin epidermal membrane from the anode to cathode when polystyrene sulfonate (PSS) oligomers are cotransported from the cathode to anode. The experiments were conducted with trace levels of the nonionic probe permeants: urea, mannitol, and raffinose. In order to systematically assess changes that occur as a result of having PSS in the cathodal chamber, the steady-state transport parameters of the membrane and the experimental permeability coefficients of the probe permeants were determined and compared with results obtained from earlier baseline experiments where both the cathodal and anodal chamber media were phosphate buffered saline. In addition, the physicochemical properties of the PSS solutions were determined including the solution viscosity and conductance as well as the mobilities of individual PSS oligomers. The effective pore radii of the transport pathways were calculated using a theoretical expression based on simultaneous diffusion and electroosmosis. Compared with the baseline results, the calculated radii were found to have increased up to around twofold and the iontophoretic fluxes of the probe permeants increased by as much sixfold. Copyright © 2011 Wiley-Liss, Inc. and the American Pharmacists Association

Development of a Methodology for the Characterisation of Air-coupled Ultrasound Probes

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

Pietroni, Paolo; Marco Revel, Gian

2010-05-28

This study is aimed at developing a technique for the characterisation of air-coupled ultrasound probes, starting from the analysis of the mechanical behaviour of the probe membrane. The vibratory behaviour of the emission membrane is studied using laser-Doppler vibrometry techniques with high frequency demodulation system (20 MHz). The determination of the vibration provides information which are useful for the assessment of the performance of the probe, in particular concerning the Quality factor and the portion of the membrane which really contributes to the emission. During the second step the results of the vibration measurements are used to calculate, by meansmore » of numerical boundary element method, the ultrasound beam emitted in terms of intensity in space. The obtained field is compared with the direct measurements carried out by scanning with the receiver probe and a pinhole plate. This comparison allows the potential and the problems of the two different characterisation techniques to be determined, even if the pinhole technique (which is currently considered the state of the art) cannot be used as an absolute reference. This study appears to be useful for paving the way for a new methodology for the calibration of air-coupled ultrasound probes, which potentially could be used not only to improve the probe manufacturing process, but also to control conformity to specifications.« less

C2 Domain of Protein Kinase Cα: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling†

PubMed Central

Kohout, Susy C.; Corbalán-García, Senena; Gómez-Fernández, Juan C.; Falke, Joseph J.

2013-01-01

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca2+-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Cα (PKCα) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca2+ binding loops and an anion binding site on β-strands 3–4, such that the β-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca2+ binding loops and the β-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCα C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca2+ activation or membrane docking trigger measurable fluorescence changes. Ca2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Φ, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca2+ first and third Ca2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on β-strands 3–4 lies near the headgroups. The data support a model in which the β-strands are tilted toward the parallel orientation relative to the membrane surface. PMID:12564928

Meyer-Overton reforged: The origins of alcohol and anesthetic potency in membranes as determined by a new NMR partitioning probe, benzyl alcohol

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

Janes, N.; Ma, L.; Hsu, J.W.

1992-01-01

The Meyer-Overton hypothesis--that anesthesia arises from the nonspecific action of solutes on membrane lipids--is reformulated using colligative thermodynamics. Configurational entropy, the randomness imparted by the solute through the partitioning process, is implicated as the energetic driving force that pertubs cooperative membrane equilibria. A proton NMR partitioning approach based on the anesthetic benzyl alcohol is developed to assess the reformulation. Ring resonances from the partitioned drug are shielded by 0.2 ppm and resolved from the free, aqueous drug. Free alcohol is quantitated in dilute lipid dispersions using an acetate internal standard. Cooperative equilibria in model dipalmitoyl lecithin membranes are examined withmore » changes in temperature and alcohol concentration. The L[sub [beta][prime

Spatial and temporal dynamics of receptor for advanced glycation endproducts, integrins, and actin cytoskeleton as probed with fluorescence-based imaging techniques

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

Syed, Aleem

Systematic spatial and temporal fluctuations are a fundamental part of any biological process. For example, lateral diffusion of membrane proteins is one of the key mechanisms in their cellular function. Lateral diffusion governs how membrane proteins interact with intracellular, transmembrane, and extracellular components to achieve their function. Herein, fluorescence-based techniques are used to elucidate the dynamics of receptor for advanced glycation end-products (RAGE) and integrin membrane proteins. RAGE is a transmembrane protein that is being used as a biomarker for various diseases. RAGE dependent signaling in numerous pathological conditions is well studied. However, RAGE lateral diffusion in the cell membranemore » is poorly understood. For this purpose, effect of cholesterol, cytoskeleton dynamics, and presence of ligand on RAGE lateral diffusion is investigated.« less

A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging

NASA Astrophysics Data System (ADS)

Tiensomjitr, Khomsan; Noorat, Rattha; Wechakorn, Kanokorn; Prabpai, Samran; Suksen, Kanoknetr; Kanjanasirirat, Phongthon; Pewkliang, Yongyut; Borwornpinyo, Suparerk; Kongsaeree, Palangpon

2017-10-01

A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of hydrazine (N2H4). In aqueous medium, the rhodol-based probe exhibited high selectivity for hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with 48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical probe also displayed a selective colorimetric response toward N2H4 from colorless solution to pink, readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300 nM (9.6 ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in live HepG2 cells by confocal fluorescence microscopy.

Surface electrostatics of lipid bilayers by EPR of a pH-sensitive spin-labeled lipid.

PubMed

Voinov, Maxim A; Rivera-Rivera, Izarys; Smirnov, Alex I

2013-01-08

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids' polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

PubMed Central

Voinov, Maxim A.; Rivera-Rivera, Izarys; Smirnov, Alex I.

2013-01-01

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes. PMID:23332063

Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria

PubMed Central

Gerencser, Akos A; Chinopoulos, Christos; Birket, Matthew J; Jastroch, Martin; Vitelli, Cathy; Nicholls, David G; Brand, Martin D

2012-01-01

Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV. PMID:22495585

Tetra(p-tolyl)borate-functionalized solvent polymeric membrane: a facile and sensitive sensing platform for peroxidase and peroxidase mimetics.

PubMed

Wang, Xuewei; Qin, Wei

2013-07-22

The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma membrane organization and dynamics is probe and cell line dependent.

PubMed

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Ultrafiltrate and microdialysis DL probe in vitro recoveries: electrolytes and metabolites

NASA Technical Reports Server (NTRS)

Janle, E. M.; Cregor, M.

1996-01-01

UF ultrafiltration and DL microdialysis probes are well-suited for sampling interstitial concentrations of ions and metabolites in peripheral tissue. The first step in utilization of membrane sampling techniques is to determine the recovery characteristics of the probes in vitro.

Fluorescent detection of apoptotic cells using a family of zinc coordination complexes with selective affinity for membrane surfaces that are enriched with phosphatidylserine.

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

Smith, Bradley D.; Lambert, Timothy N.; Lakshmi, C.

2005-03-01

The appearance of phosphatidylserine on the membrane surface of apoptotic cells (Jurkat, CHO, HeLa) is monitored by using a family of bis(Zn{sup 2+}-2,2{prime}-dipicolylamine) coordination compounds with appended fluorescein or biotin groups as reporter elements. The phosphatidylserine affinity group is also conjugated directly to a CdSe/CdS quantum dot to produce a probe suitable for prolonged observation without photobleaching. Apoptosis can be detected under a wide variety of conditions, including variations in temperature, incubation time, and binding media. Binding of each probe appears to be restricted to the cell membrane exterior, because no staining of organelles or internal membranes is observed.

A Comparison of Water Diffusion in Polymer Based Fuel Cell and Reverse Osmosis Membrane Materials

NASA Astrophysics Data System (ADS)

Soles, Christopher; Frieberg, Bradley; Tarver, Jacob; Tyagi, Madhusudan; Jeong, Cheol; Chan, Edwin; Stafford, Christopher

Hydrated polymer membranes are critical in both fuel cells and water filtration and desalination. In both of these applications the membrane function (selectively transporting or separating ions) is coupled with the transport of water through the membrane. There is a significant need to understand the nature by which the water and ions distribute and move through these membranes. This presentation compares the transport mechanisms in in an ion containing block copolymer alkaline fuel cell membrane with that of a polyamide membrane that is used as the active layer in a reverse osmosis water desalination membrane. Small angle neutron scattering measurements are used to locally probe how water swells the different materials and quantitatively describe the distribution of water within the membrane microstructures. Quasielastic neutron scattering measurements are then used to separate the polymer dynamics of the host membranes from the dynamics of the water inside the membranes. This reveals that water moves at least an order of magnitude slower through the ion containing fuel cell membrane materials, consistent with a solution-diffusion model, while the water in the polyamide membranes moves faster, consistent with a pore-flow diffusion mechanism. These insights will be discussed in terms of a coupling of the water and polymer dynamics and design cues for high performance membrane materials.

Study of rare encounters in a membrane using quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals.

PubMed

Medvedeva, Nataly; Papper, Vladislav; Likhtenshtein, Gertz I

2005-09-21

Measurements of active encounters between molecules in native membranes containing ingredients, including proteins, are of prime importance. To estimate rare encounters in a high range of rate constants (rate coefficients) and distances between interacting molecules in membranes, a cascade of photochemical reactions for molecules diffusing in multilamellar liposomes was investigated. The sensitised cascade triplet cis-trans photoisomerisation of the excited stilbene involves the use of a triplet sensitiser (Erythrosin B), a photochrome stilbene-derivative probe (4-dimethylamino-4'-aminostilbene) exhibiting the phenomenon of trans-cis photoisomerisation, and nitroxide radicals (5-doxyl stearic acid) to quench the excited triplet state of the sensitiser. Measurement of the phosphorescence lifetime of Erythrosin B and the fluorescence enhancement of the stilbene-derivative photochrome probe, at various concentrations of the nitroxide probe, made it possible to calculate the quenching rate constant k(q)= 1.1 x 10(15) cm2 M(-1) s(-1) and the rate constant of the triplet-triplet energy transfer between the sensitiser and stilbene probe k(T)= 1.0 x 10(12) cm2 M(-1) s(-1). These values, together with the data on diffusion rate constant, obtained by methods utilising various theoretical characteristic times of about seven orders of magnitude and the experimental rate constants of about five orders of magnitude, were found to be in good agreement with the advanced theory of diffusion-controlled reactions in two dimensions. Because the characteristic time of the proposed cascade method is relatively large (0.1 s), it is possible to follow rare collisions between molecules and free radicals in model and biological membranes with a very sensitive fluorescence spectroscopy technique, using a relatively low concentration of probes.

A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol

PubMed Central

Inoue, Takamasa; Tsai, Billy

2011-01-01

Non-enveloped viruses penetrate host membranes to infect cells. A cell-based assay was used to probe the endoplasmic reticulum (ER)-to-cytosol membrane transport of the non-enveloped SV40. We found that, upon ER arrival, SV40 is released into the lumen and undergoes sequential disulfide bond disruptions to reach the cytosol. However, despite these ER-dependent conformational changes, SV40 crosses the ER membrane as a large and intact particle consisting of the VP1 coat, the internal components VP2, VP3, and the genome. This large particle subsequently disassembles in the cytosol. Mutant virus and inhibitor studies demonstrate VP3 and likely the viral genome, as well as cellular proteasome, control ER-to-cytosol transport. Our results identify the sequence of events, as well as virus and host components, that regulate ER membrane penetration. They also suggest that the ER membrane supports passage of a large particle, potentially through either a sizeable protein-conducting channel or the lipid bilayer. PMID:21589906

Recording membrane potential changes through photoacoustic voltage sensitive dye

NASA Astrophysics Data System (ADS)

Zhang, Haichong K.; Kang, Jeeun; Yan, Ping; Abou, Diane S.; Le, Hanh N. D.; Thorek, Daniel L. J.; Kang, Jin U.; Gjedde, Albert; Rahmim, Arman; Wong, Dean F.; Loew, Leslie M.; Boctor, Emad M.

2017-03-01

Monitoring of the membrane potential is possible using voltage sensitive dyes (VSD), where fluorescence intensity changes in response to neuronal electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. In contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near infrared light excitation and ultrasound detection. In this work, we develop the theoretical concept whereby the voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. Based on this concept, we synthesized a novel near infrared photoacoustic VSD (PA-VSD) whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. With a 3-9 μM VSD concentration, we measured a PA signal increase in the range of 5.3 % to 18.1 %, and observed a corresponding signal reduction in fluorescence emission of 30.0 % to 48.7 %. A theoretical model successfully accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate the voltage sensing capability of the dye, but also indicate the necessity of considering both fluorescence and absorbance spectral sensitivities in order to optimize the characteristics of improved photoacoustic probes. Together, our results demonstrate photoacoustic sensing as a potential new modality for sub-second recording and external imaging of electrophysiological and neurochemical events in the brain.

Probing the Interaction between Nanoparticles and Lipid Membranes by Quartz Crystal Microbalance with Dissipation Monitoring

PubMed Central

Yousefi, Nariman; Tufenkji, Nathalie

2016-01-01

There is increasing interest in using quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the interaction of nanoparticles (NPs) with model surfaces. The high sensitivity, ease of use and the ability to monitor interactions in real-time has made it a popular technique for colloid chemists, biologists, bioengineers, and biophysicists. QCM-D has been recently used to probe the interaction of NPs with supported lipid bilayers (SLBs) as model cell membranes. The interaction of NPs with SLBs is highly influenced by the quality of the lipid bilayers. Unlike many surface sensitive techniques, by using QCM-D, the quality of SLBs can be assessed in real-time, hence QCM-D studies on SLB-NP interactions are less prone to the artifacts arising from bilayers that are not well formed. The ease of use and commercial availability of a wide range of sensor surfaces also have made QCM-D a versatile tool for studying NP interactions with lipid bilayers. In this review, we summarize the state-of-the-art on QCM-D based techniques for probing the interactions of NPs with lipid bilayers. PMID:27995125

An ESIPT fluorescent probe sensitive to protein α-helix structures.

PubMed

Jiang, Nan; Yang, Chanli; Dong, Xiongwei; Sun, Xianglang; Zhang, Dan; Liu, Changlin

2014-07-28

A large majority of membrane proteins have one or more transmembrane regions consisting of α-helices. Membrane protein levels differ from one type of cell to another, and the expression of membrane proteins also changes from normal to diseased cells. For example, prostate cancer cells have been reported to have downregulated expression of membrane proteins, including zinc transporters, compared with normal prostate cells. These reports inspired us to design a fluorescence probe sensitive to protein α-helical structures to discriminate individual prostate cancer cells from normal ones. A benzazole derivative ( in this study) was observed to emit strong fluorescence resulting from an excited-state intramolecular proton transfer (ESIPT) in protein α-helical environments. The intensity of ESIPT fluorescence of was observed to be positively correlated with the α-helix content of proteins. The molecular docking simulation suggested that it had low energy for the binding of to proteins when the binding sites were localized within the α-helical regions of protein via H-bonds. Furthermore, was found to be localized in cell membranes through binding to transmembrane α-helical regions of membrane proteins, and was capable of probing differences in the α-helix contents of membrane proteins between normal and cancerous prostate cells through changes in the ESIPT emission intensity. These results indicated that could distinguish individual prostate cancer cells from normal ones, as the changes in the ESIPT fluorescence intensity of could reflect the regulation in expression of the membrane proteins including zinc transporters. This recognition strategy of individual prostate cancer cells might contribute to early diagnosis techniques for prostate cancer.

Plant Available Nutrients, Barrow, Alaska, Ver. 1

DOE Data Explorer

Sloan, Victoria; Liebig, Jenny; Curtis, Bryan; Hahn, Melanie; Iversen, Colleen; Siegrist, Julie

2014-02-19

This dataset consists of measurements of plant available nutrients made using Plant Root Simulator probes (Western Ag Innovations Inc.) during 2012 and 2013. In 2012, Ca, Mg, K, P, Fe, Mn, Cu, Zn, B, S, Pb, Al, Cd, NO3-N and NH4-N were measured during spring, summer and winter in the centers, edges and troughs of four polygons in each of four areas of contrasting moisture regime and polygon type. In 2013, probes were installed in centers, edges and troughs of four polygons in each of two areas (high-centered and low-centered polygons) at two-week intervals and at 3 soil depths to capture fine-scale season dynamics of NO3-N and NH4-N. PRS probes are ion exchange resin membranes held in plastic supports that are inserted into soil to measure ion supply in situ. The anion and cation exchange with the membrane is intended to mimic plant uptake and thus provide a relevant measure of soil nutrient bioavailability. Measurements are made per area of probe membrane and cannot be converted to concentrations or related to soil volume.

A Ratiometric Two-Photon Fluorescent Probe for Tracking the Lysosomal ATP Level: Direct in cellulo Observation of Lysosomal Membrane Fusion Processes.

PubMed

Jun, Yong Woong; Wang, Taejun; Hwang, Sekyu; Kim, Dokyoung; Ma, Donghee; Kim, Ki Hean; Kim, Sungjee; Jung, Junyang; Ahn, Kyo Han

2018-06-05

Vesicles exchange its contents through membrane fusion processes-kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We disclose a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging technique, lysosomal membrane fusion process in cells has been directly observed along with the concentration of its content-lysosomal ATP. Results show that the kiss-and-run process between lysosomes proceeds through repeating transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanopillar arrays on semiconductor membranes as electron emission amplifiers.

PubMed

Qin, Hua; Kim, Hyun-Seok; Blick, Robert H

2008-03-05

A new transmission-type electron multiplier was fabricated from silicon-on-insulator (SOI) material by integrating an array of one-dimensional (1D) silicon nanopillars onto a two-dimensional (2D) silicon membrane. Primary electrons are injected into the nanopillar-membrane (NPM) system from the flat surface of the membrane, while electron emission from the nanopillars is probed by an anode. The secondary electron yield (SEY) from the nanopillars in the current device is found to be about 1.8 times that of the plain silicon membrane. This gain in electron number is slightly enhanced by the electric field applied from the anode. Further optimization of the dimensions of the NPM and an application of field emission promise an even higher gain for detector applications and allow for probing of electronic/mechanical excitations in an NPM system stimulated by incident particles or radiation.

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

PubMed

Elías-Wolff, Federico; Lindén, Martin; Lyubartsev, Alexander P; Brandt, Erik G

2018-03-13

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

A fixable probe for visualizing flagella and plasma membranes of the African trypanosome.

PubMed

Wiedeman, Justin; Mensa-Wilmot, Kojo

2018-01-01

The protozoan Trypanosoma brucei sp. cause diseases in humans and animals. Studies of T. brucei cell biology have revealed unique features, such as major endocytic events being limited to a single region, and mitochondrial genome segregation mediated via basal bodies. Further understanding of trypanosome cell biology can be facilitated with super-resolution fluorescence microscopy. Lack of a plasma membrane probe for fixed trypanosomes remains a persistent problem in need of a working solution. Herein, we report protocols developed using mCLING in super-resolution structured illumination fluorescence microscopy (SR-SIM). mCLING comprehensively labels flagellar membranes, including nascent intracellular stages. To extend its usefulness for trypanosome biology we optimized mCLING in combination with organelle-specific antibodies for immunofluorescence of basal bodies or mitochondria. Then in work with live trypanosomes, we demonstrated internalization of mCLING into endocytic stations that overlap with LysoTracker in acidic organelles. Greater detail of the intracellular location of mCLING was obtained with SR-SIM after pulsing trypanosomes with the probe, and allowing continuous uptake of fluorescent concanavalin A (ConA) destined for lysosomes. In most cases, ConA and mCLING vesicles were juxtaposed but not coincident. A video of the complete image stack at the 15 min time point shows zones of mCLING staining surrounding patches of ConA, consistent with persistence of mCLING in membranes of compartments that contain luminal ConA. In summary, these studies establish mCLING as a versatile trypanosome membrane probe compatible with super-resolution microscopy that can be used for detailed analysis of flagellar membrane biogenesis. In addition, mCLING can be used for immunofluorescence in fixed, permeabilized trypanosomes. Its robust staining of the plasma membrane eliminates a need to overlay transmitted light images on fluorescence pictures obtained from widefield, confocal, or super-resolution microscopy.

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation.

PubMed

Carquin, Mélanie; Conrard, Louise; Pollet, Hélène; Van Der Smissen, Patrick; Cominelli, Antoine; Veiga-da-Cunha, Maria; Courtoy, Pierre J; Tyteca, Donatienne

2015-12-01

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

A cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing.

PubMed

Ke, Guoliang; Zhu, Zhi; Wang, Wei; Zou, Yuan; Guan, Zhichao; Jia, Shasha; Zhang, Huimin; Wu, Xuemeng; Yang, Chaoyong James

2014-09-10

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid-DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid-DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid-DNA probe showed sensitive and reversible response to pH change in the range of 6.0-8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid-DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid-DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

1995-11-28

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

1995-01-01

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

Diffusion studies on permeable nitroxyl spin probes through bilayer lipid membranes: A low frequency ESR study

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

Meenakumari, V.; Benial, A. Milton Franklin, E-mail: miltonfranklin@yahoo.com; Utsumi, Hideo

2015-06-24

Electron spin resonance (ESR) studies were carried out for permeable 2mM {sup 14}N-labeled deutrated 3 Methoxy carbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) in pure water and 1mM, 2mM, 3mM, 4mM concentration of 14N-labeled deutrated MC-PROXYL in 400mM concentration of liposomal solution by using a 300 MHz ESR spectrometer. The ESR parameters such as linewidth, hyperfine coupling constant, g-factor, partition parameter and permeability were reported for these samples. The line broadening was observed for the nitroxyl spin probe in the liposomal solution. The line broadening indicates that the high viscous nature of the liposomal solution. The partition parameter and permeability values indicate the maximum diffusion ofmore » nitroxyl spin probes in the bilayer lipid membranes at 2 mM concentration of nitroxyl radical. This study illustrates that ESR can be used to differentiate between the intra and extra- membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe. From the ESR results, the spin probe concentration was optimized as 2mM in liposomal solution for ESR phantom studies/imaging, invivo and invitro experiments.« less

Monitoring ion-channel function in real time through quantum decoherence

PubMed Central

Hall, Liam T.; Hill, Charles D.; Cole, Jared H.; Städler, Brigitte; Caruso, Frank; Mulvaney, Paul; Wrachtrup, Jörg; Hollenberg, Lloyd C. L.

2010-01-01

In drug discovery, there is a clear and urgent need for detection of cell-membrane ion-channel operation with wide-field capability. Existing techniques are generally invasive or require specialized nanostructures. We show that quantum nanotechnology could provide a solution. The nitrogen-vacancy (NV) center in nanodiamond is of great interest as a single-atom quantum probe for nanoscale processes. However, until now nothing was known about the quantum behavior of a NV probe in a complex biological environment. We explore the quantum dynamics of a NV probe in proximity to the ion channel, lipid bilayer, and surrounding aqueous environment. Our theoretical results indicate that real-time detection of ion-channel operation at millisecond resolution is possible by directly monitoring the quantum decoherence of the NV probe. With the potential to scan and scale up to an array-based system, this conclusion may have wide-ranging implications for nanoscale biology and drug discovery. PMID:20937908

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors.

PubMed

Yi, Hyunjung; Ghosh, Debadyuti; Ham, Moon-Ho; Qi, Jifa; Barone, Paul W; Strano, Michael S; Belcher, Angela M

2012-03-14

Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. © 2012 American Chemical Society

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors

PubMed Central

HAM, MOON-HO; QI, JIFA; BARONE, PAUL W.; STRANO, MICHAEL S.; BELCHER, ANGELA M.

2014-01-01

Second near-infrared (NIR) window light (950-1,400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to four-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control non-targeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. PMID:22268625

Monitoring ion-channel function in real time through quantum decoherence.

PubMed

Hall, Liam T; Hill, Charles D; Cole, Jared H; Städler, Brigitte; Caruso, Frank; Mulvaney, Paul; Wrachtrup, Jörg; Hollenberg, Lloyd C L

2010-11-02

In drug discovery, there is a clear and urgent need for detection of cell-membrane ion-channel operation with wide-field capability. Existing techniques are generally invasive or require specialized nanostructures. We show that quantum nanotechnology could provide a solution. The nitrogen-vacancy (NV) center in nanodiamond is of great interest as a single-atom quantum probe for nanoscale processes. However, until now nothing was known about the quantum behavior of a NV probe in a complex biological environment. We explore the quantum dynamics of a NV probe in proximity to the ion channel, lipid bilayer, and surrounding aqueous environment. Our theoretical results indicate that real-time detection of ion-channel operation at millisecond resolution is possible by directly monitoring the quantum decoherence of the NV probe. With the potential to scan and scale up to an array-based system, this conclusion may have wide-ranging implications for nanoscale biology and drug discovery.

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

PubMed Central

Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

2002-01-01

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress

PubMed Central

SWIFT, LUTHER M.; SARVAZYAN, NARINE

2011-01-01

Localization and staining features of the oxidant-sensitive fluorescent probe 2 7 - dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria. PMID:10710368

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography

PubMed Central

Page, Michael J.; Lourenço, André L.; David, Tovo; LeBeau, Aaron M.; Cattaruzza, Fiore; Castro, Helena C.; VanBrocklin, Henry F.; Coughlin, Shaun R.; Craik, Charles S.

2015-01-01

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements. PMID:26423607

Disruption of gel phase lipid packing efficiency by sucralose studied with merocyanine 540.

PubMed

Barker, Morgan; Kennedy, Anthony

2017-04-01

Sucralose, an artificial sweetener, displays very different behavior towards membranes than its synthetic precursor sucrose. The impact of both sugars on model dipalmitoylphosphatidylcholine model membranes was investigated using absorbance and flourescence spectroscopy and the membrane probe merocyanine 540. This probe molecule is highly sensitive to changes in membrane packing, microviscosity and polarity. This work focuses on the impact of sugars on the outer leaflet of unilamellar dipalmitoyl phosphatidylcholine model membranes. The choice of lipid permits access to the gel phase at room temperature and incorporation of the dye after liposome formation allows us to examine the direct impact of the sugar on the outer leaflet while maximizing the response of the dye to changes in the bilayer. The results demonstrate that sucrose has no impact on the packing efficiency of lipids in unilamellar DPPC vesicles in the gel phase. Conversely sucralose decreases the packing efficiency of lipids in the gel phase and results in decreased microviscosity and increased membrane fluidity, which may be as a result of water disruption at the membrane water interface. Copyright © 2017 Elsevier B.V. All rights reserved.

Membrane Orientation and Lateral Diffusion of BODIPY-Cholesterol as a Function of Probe Structure

PubMed Central

Solanko, Lukasz M.; Honigmann, Alf; Midtiby, Henrik Skov; Lund, Frederik W.; Brewer, Jonathan R.; Dekaris, Vjekoslav; Bittman, Robert; Eggeling, Christian; Wüstner, Daniel

2013-01-01

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells. PMID:24209853

Nanoscale patterning controls inorganic-membrane interface structure

NASA Astrophysics Data System (ADS)

Almquist, Benjamin D.; Verma, Piyush; Cai, Wei; Melosh, Nicholas A.

2011-02-01

The ability to non-destructively integrate inorganic structures into or through biological membranes is essential to realizing full bio-inorganic integration, including arrayed on-chip patch-clamps, drug delivery, and biosensors. Here we explore the role of nanoscale patterning on the strength of biomembrane-inorganic interfaces. AFM measurements show that inorganic probes functionalized with hydrophobic bands with thicknesses complimentary to the hydrophobic lipid bilayer core exhibit strong attachment in the bilayer. As hydrophobic band thickness increases to 2-3 times the bilayer core the interfacial strength decreases, comparable to homogeneously hydrophobic probes. Analytical calculations and molecular dynamics simulations predict a transition between a `fused' interface and a `T-junction' that matches the experimental results, showing lipid disorder and defect formation for thicker bands. These results show that matching biological length scales leads to more intimate bio-inorganic junctions, enabling rational design of non-destructive membrane interfaces.The ability to non-destructively integrate inorganic structures into or through biological membranes is essential to realizing full bio-inorganic integration, including arrayed on-chip patch-clamps, drug delivery, and biosensors. Here we explore the role of nanoscale patterning on the strength of biomembrane-inorganic interfaces. AFM measurements show that inorganic probes functionalized with hydrophobic bands with thicknesses complimentary to the hydrophobic lipid bilayer core exhibit strong attachment in the bilayer. As hydrophobic band thickness increases to 2-3 times the bilayer core the interfacial strength decreases, comparable to homogeneously hydrophobic probes. Analytical calculations and molecular dynamics simulations predict a transition between a `fused' interface and a `T-junction' that matches the experimental results, showing lipid disorder and defect formation for thicker bands. These results show that matching biological length scales leads to more intimate bio-inorganic junctions, enabling rational design of non-destructive membrane interfaces. Electronic supplementary information (ESI) available: Breakthrough rate as a function of force plots for 5 nm, 10 nm and ∞-probes.. See DOI: 10.1039/c0nr00486c

Ratiometric fluorescence measurements and imaging of the dipole potential in cell plasma membranes

NASA Astrophysics Data System (ADS)

Shynkar, Vasyl V.; Klymchenko, Andrey S.; Duportail, Guy; Demchenko, Alexander P.; Mély, Yves

2004-09-01

Development of fluorescence microscopic methods is limited by the application of new dyes, the response of which could be sensitive to different functional states in the living cells, and, in particular, to electrostatic potentials on their plasma membranes. Recently, we showed that newly designed 3-hydroxyflavone fluorescence dyes are highly electrochromic and show a strong two-band ratiometric response to electric dipole potential in lipid membranes. In the present report we extend these observations and describe a new generation of these dyes as electrochromic probes in biomembrane research. Modification of the membrane dipole potential was achieved by addition of 6-ketocholestanol (6-KC), cholesterol and phloretin. The dipole potential was also estimated by the reference probe di-8-ANEPPS. As an example, we show that on addition of 6-KC there occurs a dramatic change of the intensity ratio of the two emission bands, which is easily detected as a change of color. We describe in detail the applications of one of these dyes, PPZ8, to the studies of cells in suspension or attached to the glass surface. Confocal microscopy demonstrates strong preference of the probe for the cell plasma membrane, which allows us to apply this dye for studying electrostatic and other biomembrane properties. We demonstrate that the two-color response provides a direct and convenient way to measure the dipole potential in the plasma membrane. Applying PPZ8 in confocal microcopy and two-photon microspectroscopy allowed us to provide two-color imaging of the membrane dipole potential on the level of a single cell.

Effects of pipette modulation and imaging distances on ion currents measured with scanning ion conductance microscopy (SICM).

PubMed

Chen, Chiao-Chen; Baker, Lane A

2011-01-07

Local conductance variations can be estimated by measuring ion current magnitudes with scanning ion conductance microscopy (SICM). Factors which influence image quality and quantitation of ion currents measured with SICM have been evaluated. Specifically, effects of probe-sample separation and pipette modulation have been systematically studied for the case of imaging conductance variations at pores in a polymer membrane under transmembrane concentration gradients. The influence of probe-sample separation on ion current images was evaluated using distance-modulated (ac) feedback. Approach curves obtained using non-modulated (dc) feedback were also recorded to determine the relative influence of pipette-generated convection by comparison of ion currents measured with both ac and dc feedback modes. To better interpret results obtained, comparison to a model based on a disk-shaped geometry for nanopores in the membrane, as well as relevant position-dependent parameters of the experiment is described. These results advance our current understanding of conductance measurements with SICM.

Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration

PubMed Central

Brugués, Jan; Maugis, Benoit; Casademunt, Jaume; Nassoy, Pierre; Amblard, François; Sens, Pierre

2010-01-01

Bleb-based cell motility proceeds by the successive inflation and retraction of large spherical membrane protrusions (“blebs”) coupled with substrate adhesion. In addition to their role in motility, cellular blebs constitute a remarkable illustration of the dynamical interactions between the cytoskeletal cortex and the plasma membrane. Here we study the bleb-based motions of Entamoeba histolytica in the constrained geometry of a micropipette. We construct a generic theoretical model that combines the polymerization of an actin cortex underneath the plasma membrane with the myosin-generated contractile stress in the cortex and the stress-induced failure of membrane-cortex adhesion. One major parameter dictating the cell response to micropipette suction is the stationary cortex thickness, controlled by actin polymerization and depolymerization. The other relevant physical parameters can be combined into two characteristic cortex thicknesses for which the myosin stress (i) balances the suction pressure and (ii) provokes membrane-cortex unbinding. We propose a general phase diagram for cell motions inside a micropipette by comparing these three thicknesses. In particular, we theoretically predict and experimentally verify the existence of saltatory and oscillatory motions for a well-defined range of micropipette suction pressures. PMID:20713731

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

PubMed

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Spectroscopic probing of ortho-nitrophenol localization in phospholipid bilayers.

PubMed

Sánchez, Julieta M; Turina, Anahí del V; Perillo, María A

2007-11-12

In the present study we estimated the localization of ortho-nitrophenol (ONP) within model membranes through its efficiency to quench and to modify the anisotropy of DPH and TMA-DPH fluorescence. These fluorescent probes are known to sense the hydrocarbon core and the polar head group region of membranes, respectively. TMA-DPH fluorescence in MLVs was more efficiently quenched than DPH (K(q,TMA-DPH)=2.36 and K(q,DPH)=1.07 mM ns(-1)). Moreover, these results demonstrated the interfacial localization of ONP and may contribute to understand membrane-mediated mechanisms of ONP-induced toxicity and the behavior of ONP as a product of several enzymatic reactions occurring in the presence of lipid-water interfaces.

Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

PubMed Central

Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

2012-01-01

A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

Fluorescent Probes for Sensitive and Selective Detection of pH Changes in Live Cells in Visible and Near-infrared Channels.

PubMed

Fang, Mingxi; Adhikari, Rashmi; Bi, Jianheng; Mazi, Wafa; Dorh, Nethaniah; Wang, Jianbo; Conner, Nathan; Ainsley, Jon; Karabencheva-Christova, Tatyana G; Luo, Fen-Tair; Tiwari, Ashutosh; Liu, Haiying

2017-12-28

We report five fluorescent probes based on coumarin-hybridized fluorescent dyes with spirolactam ring structures (A-E) to detect pH changes in live cell by monitoring visible and near-infrared fluorescence changes. Under physiological or basic conditions, the fluorescent probes A, B, C, D and E preserve their spirolactam ring-closed forms and only display fluorescent peaks in the visible region corresponding to coumarin moieties at 497, 483, 498, 497 and 482 nm, respectively. However, at acidic pH, the rings of the spirolactam forms of the fluorescent probes A, B, C, D and E open up, generating new near-infrared fluorescence peaks at 711, 696, 707, 715, and 697 nm, respectively, through significantly extended π-conjugation to coumarin moieties of the fluorophores. The fluorescent probes B and E can be applied to visualize pH changes by monitoring visible as well as near-infrared fluorescence changes. This helps avoid fluorescence imaging blind spots at neutral or basic pH, which typical pH fluorescent probes encounter. The probes exhibit high sensitivity to pH changes, excellent photostability, low auto-fluorescence background and good cell membrane permeability.

PEGylated Red-Emitting Calcium Probe with Improved Sensing Properties for Neuroscience.

PubMed

Ponsot, Flavien; Shen, Weida; Ashokkumar, Pichandi; Audinat, Etienne; Klymchenko, Andrey S; Collot, Mayeul

2017-11-22

Monitoring calcium concentration in the cytosol is of main importance as this ion drives many biological cascades within the cell. To this end, molecular calcium probes are widely used. Most of them, especially the red emitting probes, suffer from nonspecific interactions with inner membranes due to the hydrophobic nature of their fluorophore. To circumvent this issue, calcium probes conjugated to dextran can be used to enhance the hydrophilicity and reduce the nonspecific interaction and compartmentalization. However, dextran conjugates also feature important drawbacks including lower affinity, lower dynamic range, and slow diffusion. Herein, we combined the advantage of molecular probes and dextran conjugate without their drawbacks by designing a new red emitting turn-on calcium probe based on PET quenching, Rhod-PEG, in which the rhodamine fluorophore bears four PEG 4 units. This modification led to a high affinity calcium probe (K d = 748 nM) with reduced nonspecific interactions, enhanced photostability, two-photon absorbance, and brightness compared to the commercially available Rhod-2. After spectral characterizations, we showed that Rhod-PEG quickly and efficiently diffused through the dendrites of pyramidal neurons with an enhanced sensitivity (ΔF/F 0 ) at shorter time after patching compared to Rhod-2.

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe.

PubMed

Shvadchak, Volodymyr V; Falomir-Lockhart, Lisandro J; Yushchenko, Dmytro A; Jovin, Thomas M

2011-04-15

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.

Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

PubMed

Smertenko, Andrei; Moschou, Panagiotis; Zhang, Laining; Fahy, Deirdre; Bozhkov, Peter

2016-01-01

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

Nanobits, Nembranes and Micro Four-Point Probes: Customizable Tools for insitu Manipulation and Characterisation of Nanostructures

NASA Astrophysics Data System (ADS)

Boggild, Peter; Hjorth Petersen, Dirch; Sardan Sukas, Ozlem; Dam, Henrik Friis; Lei, Anders; Booth, Timothy; Molhave, Kristian; Eicchorn, Volkmar

2010-03-01

We present a range of highly adaptable microtools for direct interaction with nanoscale structures; (i) semiautomatic pick-and-place assembly of multiwalled carbon nanotubes onto cantilevers for high-aspect ratio scanning probe microscopy, using electrothermal microgrippers inside a SEM. Topology optimisation was used to calculate the optimal gripper shape defined by the boundary conditions, resulting in 10-100 times better performance. By instead pre-defining detachable tips using electron beam lithography, free-form scanning probe tips (Nanobits) can be mounted in virtually any position on a cantilever; (ii) scanning micro four point probes allow fast, non- destructive mapping of local electrical properties (sheet resistance and Hall mobility) and hysteresis effects of graphene sheets; (iii) sub 100 nm freestanding devices with wires, heaters, actuators, sensors, resonators and probes were defined in a 100 nm thin membrane with focused ion beam milling. By patterning generic membrane templates (Nembranes) the fabrication time of a TEM compatible NEMS device is effectively reduced to less around 20 minutes.

The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

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

Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.

2010-12-17

Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended themore » study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.« less

Structured Water Layers Adjacent to Biological Membranes

PubMed Central

Higgins, Michael J.; Polcik, Martin; Fukuma, Takeshi; Sader, John E.; Nakayama, Yoshikazu; Jarvis, Suzanne P.

2006-01-01

Water amid the restricted space of crowded biological macromolecules and at membrane interfaces is essential for cell function, though the structure and function of this “biological water” itself remains poorly defined. The force required to remove strongly bound water is referred to as the hydration force and due to its widespread importance, it has been studied in numerous systems. Here, by using a highly sensitive dynamic atomic force microscope technique in conjunction with a carbon nanotube probe, we reveal a hydration force with an oscillatory profile that reflects the removal of up to five structured water layers from between the probe and biological membrane surface. Further, we find that the hydration force can be modified by changing the membrane fluidity. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine gel (Lβ) phase bilayers, each oscillation in the force profile indicates the force required to displace a single layer of water molecules from between the probe and bilayer. In contrast, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine fluid (Lα) phase bilayers at 60°C and 1,2-dioleoyl-sn-glycero-3-phosphocholine fluid (Lα) phase bilayers at 24°C seriously disrupt the molecular ordering of the water and result predominantly in a monotonic force profile. PMID:16798815

Use of unnatural amino acids to probe structure-activity relationships and mode-of-action of antimicrobial peptides.

PubMed

Tossi, Alessandro; Scocchi, Marco; Zahariev, Sotir; Gennaro, Renato

2012-01-01

Endogenous antimicrobial peptides (AMPs) can have multimodal mechanisms of bacterial inactivation, such as membrane lysis, interference with cell wall biosynthesis or membrane-based protein machineries, or translocation through the membrane to intracellular targets. The controlled variation of side-chain characteristics in their amino acid residues can provide much useful information on structure-activity relationships and mode-of-action, and also lead to improved activities. The small size and relatively low complexity of AMPs make them amenable to solid-phase peptide synthesis, facilitating the use of nonproteinogenic amino acids and vastly increasing the accessible molecular diversity of side chains. Here, we describe how such residues can be used to modulate such key parameters as cationicity, hydrophobicity, steric factors conformational stability, and H-bonding.

Water Dynamics in Nafion Fuel Cell Membranes: the Effects of Confinement and Structural Changes on the Hydrogen Bond Network

PubMed Central

Moilanen, David E.; Piletic, Ivan R.; Fayer, Michael D.

2008-01-01

The complex environments experienced by water molecules in the hydrophilic channels of Nafion membranes are studied by ultrafast infrared pump-probe spectroscopy. A wavelength dependent study of the vibrational lifetime of the O-D stretch of dilute HOD in H2O confined in Nafion membranes provides evidence of two distinct ensembles of water molecules. While only two ensembles are present at each level of membrane hydration studied, the characteristics of the two ensembles change as the water content of the membrane changes. Time dependent anisotropy measurements show that the orientational motions of water molecules in Nafion membranes are significantly slower than in bulk water and that lower hydration levels result in slower orientational relaxation. Initial wavelength dependent results for the anisotropy show no clear variation in the time scale for orientational motion across a broad range of frequencies. The anisotropy decay is analyzed using a model based on restricted orientational diffusion within a hydrogen bond configuration followed by total reorientation through jump diffusion. PMID:18728757

Probing eukaryotic cell mechanics via mesoscopic simulations

NASA Astrophysics Data System (ADS)

Pivkin, Igor V.; Lykov, Kirill; Nematbakhsh, Yasaman; Shang, Menglin; Lim, Chwee Teck

2017-11-01

We developed a new mesoscopic particle based eukaryotic cell model which takes into account cell membrane, cytoskeleton and nucleus. The breast epithelial cells were used in our studies. To estimate the viscoelastic properties of cells and to calibrate the computational model, we performed micropipette aspiration experiments. The model was then validated using data from microfluidic experiments. Using the validated model, we probed contributions of sub-cellular components to whole cell mechanics in micropipette aspiration and microfluidics experiments. We believe that the new model will allow to study in silico numerous problems in the context of cell biomechanics in flows in complex domains, such as capillary networks and microfluidic devices.

Visual detection of copper(II) ions in blood samples by controlling the leaching of protein-capped gold nanoparticles.

PubMed

Lee, Yen-Fei; Deng, Ting-Wei; Chiu, Wei-Jane; Wei, Tsao-Yen; Roy, Prathik; Huang, Chih-Ching

2012-04-21

We have developed a simple, low-cost, paper-based probe for the selective colorimetric detection of copper ions (Cu(2+)) in aqueous solutions. The bovine serum albumin (BSA)-modified 13.3-nm Au nanoparticle (BSA-Au NP) probe was designed to detect Cu(2+) ions using lead ions (Pb(2+)) and 2-mercaptoethanol (2-ME) as leaching agents in a glycine-NaOH (pH 12.0) solution. In addition, a nitrocellulose membrane (NCM) was used to trap the BSA-Au NPs, leading to the preparation of a nanocomposite film consisting of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs probe operates on the principle that Cu deposition on the surface of the BSA-Au NPs inhibits their leaching ability, which is accelerated by Pb(2+) ions in the presence of 2-ME. Under optimal solution conditions (5 mM glycine-NaOH (pH 12.0), Pb(2+) (50 μM), and 2-ME (1.0 M)), the Pb(2+)/2-ME-BSA-Au NPs/NCM enabled the detection of Cu(2+) at nanomolar concentrations in aqueous solutions by the naked eye with high selectivity (at least 100-fold over other metal ions). In addition, this cost-effective probe allowed for the rapid and simple determination of Cu(2+) ions in not only natural water samples but also in a complex biological sample (in this case, blood sample).

Neutron scattering to study membrane systems: from lipid vesicles to living cells.

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

Nickels, Jonathan D.; Chatterjee, Sneha; Stanley, Christopher B.

The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent workmore » using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.« less

Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure.

PubMed

Solanko, Lukasz M; Honigmann, Alf; Midtiby, Henrik Skov; Lund, Frederik W; Brewer, Jonathan R; Dekaris, Vjekoslav; Bittman, Robert; Eggeling, Christian; Wüstner, Daniel

2013-11-05

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Paper membrane-based SERS platform for the determination of glucose in blood samples.

PubMed

Torul, Hilal; Çiftçi, Hakan; Çetin, Demet; Suludere, Zekiye; Boyacı, Ismail Hakkı; Tamer, Uğur

2015-11-01

In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications.

A chromogenic and fluorogenic rhodol-based chemosensor for hydrazine detection and its application in live cell bioimaging

NASA Astrophysics Data System (ADS)

Tiensomjitr, Khomsan; Noorat, Rattha; Chomngam, Sinchai; Wechakorn, Kanokorn; Prabpai, Samran; Kanjanasirirat, Phongthon; Pewkliang, Yongyut; Borwornpinyo, Suparerk; Kongsaeree, Palangpon

2018-04-01

A rhodol-based fluorescent probe has been developed as a selective hydrazine chemosensor using levulinate as a recognition site. The rhodol levulinate probe (RL) demonstrated high selectivity and sensitivity toward hydrazine among other molecules. The chromogenic response of RL solution to hydrazine from colorless to pink could be readily observed by the naked eye, while strong fluorescence emission could be monitored upon excitation at 525 nm. The detection process occurred via a ring-opening process of the spirolactone initiated by hydrazinolysis, triggering the fluorescence emission with a 53-fold enhancement. The probe rapidly reacted with hydrazine in aqueous medium with the detection limit of 26 nM (0.83 ppb), lower than the threshold limit value (TLV) of 10 ppb suggested by the U.S. Environmental Protection Agency. Furthermore, RL-impregnated paper strips could detect hydrazine vapor. For biological applicability of RL, its membrane-permeable property led to bioimaging of hydrazine in live HepG2 cells by confocal fluorescence microscopy.

Imaging and Detection of Carboxylesterase in Living Cells and Zebrafish Pretreated with Pesticides by a New Near-Infrared Fluorescence Off-On Probe.

PubMed

Li, Dongyu; Li, Zhao; Chen, Weihua; Yang, Xingbin

2017-05-24

A new near-infrared fluorescence off-on probe was developed and applied to fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid). The probe was readily prepared by connecting (4-acetoxybenzyl)oxy as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR-780. The fluorescence off-on response of the probe to carboxylesterase is based on the enzyme-catalyzed spontaneous hydrolysis of the carboxylic ester bond, followed by a further fragmentation of the phenylmethyl unit and thereby the fluorophore release. Compared with the only existing near-infrared carboxylesterase probe, the proposed probe exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 4.5 × 10 -3 U/mL. More importantly, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for monitoring carboxylesterase activity in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure.

Design and fabrication of nanoelectrodes for applications with scanning electrochemical microscopy

NASA Astrophysics Data System (ADS)

Thakar, Rahul

Scanning electrochemical microscope (SECM) was introduced two decades ago and has since emerged as a powerful research tool to investigate localized electrochemical reactions at the surface of material and biological samples. The ability to obtain chemical information at a surface differentiates SECM from competing scanning probe microscopy (SPM) techniques. Although, chemical specificity is a unique advantage offered by SECM, inherent limitations due to a slow feedback response, and challenges associated with production of smaller electrodes have remained major drawbacks. Initially in this research, SECM was utilized as a characterization and investigative tool. Later, advances in SECM imaging were achieved with design and production of multifunctional nanoelectrodes. At first, platinum based nanoelectrodes were fabricated for use as electrochemical probes to investigate local electron transfer at chemically-modified surfaces. Further, micron and sub-micron platinum electrodes with chemically modified shrouds were prepared and characterized with voltammetric measurements. Studies reveal experimental evidence for the presence of edge-effects that are typically associated with submicron electrodes. Interestingly, we observed selectivity of these electrodes based on hydrophobic/ hydrophilic character. Through vapor deposition of parylene over microstructured material, single-pore membranes and porous membrane arrays were produced. Pore size characterization within porous membranes was performed with templated growth of micro/nanostructures. Characterization of transport properties of ions and redox-active molecules through hydrophobic parylene membranes was investigated with ion conductance microscopy and SECM, individually. Parylene is an insulative material that is chemically resistant, deposits conformally over high-aspect ratio objects and also converts into conductive carbon at high-temperature pyrolysis. Motivated by these results we identified a unique strategy to fabricate parylene based carbon electrodes Here, we have developed a unique strategy to obtain carbon based nanoelectrodes from vapor deposition of parylene over pulled glass nanopipettes. With this approach, multiple electrode geometries were constructed and the application of individual geomtery with SECM is demonstrated. In particular, enhanced spatial resolution and electrochemical information were obtained with the use of carbon ring/nanopore electrodes. Practical implications of edge-effects observed with carbon ring/nanopore electrodes is discussed with substrate generation tip collection (SG/TC) SECM Carbon ring/nanopore electrodes have also enabled the use of SECM in conjunction with ion conductance microscopy to alleviate the issue of poor feedback response. This has further helped in deconvolution of electrochemcial and topographical signals. Although, use of carbon nanoelectrodes is discussed with specific applications to electrochemcial microscopy, these probes have wide utility in electroanalytical applications. Initial proof-of-concept experiments along with future directions for this work are presented.

Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids in model membranes is connected not to lipid mobility but to probe location.

PubMed

Amaro, Mariana; Filipe, Hugo A L; Prates Ramalho, J P; Hof, Martin; Loura, Luís M S

2016-03-14

Nitrobenzoxadiazole (NBD)-labeled lipids are popular fluorescent membrane probes. However, the understanding of important aspects of the photophysics of NBD remains incomplete, including the observed shift in the emission spectrum of NBD-lipids to longer wavelengths following excitation at the red edge of the absorption spectrum (red-edge excitation shift or REES). REES of NBD-lipids in membrane environments has been previously interpreted as reflecting restricted mobility of solvent surrounding the fluorophore. However, this requires a large change in the dipole moment (Δμ) of NBD upon excitation. Previous calculations of the value of Δμ of NBD in the literature have been carried out using outdated semi-empirical methods, leading to conflicting values. Using up-to-date density functional theory methods, we recalculated the value of Δμ and verified that it is rather small (∼2 D). Fluorescence measurements confirmed that the value of REES is ∼16 nm for 1,2-dioleoyl-sn-glycero-3-phospho-l-serine-N-(NBD) (NBD-PS) in dioleoylphosphatidylcholine vesicles. However, the observed shift is independent of both the temperature and the presence of cholesterol and is therefore insensitive to the mobility and hydration of the membrane. Moreover, red-edge excitation leads to an increased contribution of the decay component with a shorter lifetime, whereas time-resolved emission spectra of NBD-PS displayed an atypical blue shift following excitation. This excludes restrictions to solvent relaxation as the cause of the measured REES and TRES of NBD, pointing instead to the heterogeneous transverse location of probes as the origin of these effects. The latter hypothesis was confirmed by molecular dynamics simulations, from which the calculated heterogeneity of the hydration and location of NBD correlated with the measured fluorescence lifetimes/REES. Globally, our combination of theoretical and experiment-based techniques has led to a considerably improved understanding of the photophysics of NBD and a reinterpretation of its REES in particular.

Structural changes in lymphocytes membrane of Chernobyl clean-up workers from Latvia.

PubMed

Kalnina, Inta; Zvagule, Tija; Gabruseva, Natalija; Kirilova, Jelena; Kurjane, Natalja; Bruvere, Ruta; Kesters, Andris; Kizane, Gunta; Kirilovs, Georgijs; Meirovics, Imants

2007-11-01

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies . The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997-2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.

Development of a DNA Sensor Based on Nanoporous Pt-Rich Electrodes

NASA Astrophysics Data System (ADS)

Van Hao, Pham; Thanh, Pham Duc; Xuan, Chu Thi; Hai, Nguyen Hoang; Tuan, Mai Anh

2017-06-01

Nanoporous Pt-rich electrodes with 72 at.% Pt composition were fabricated by sputtering a Pt-Ag alloy, followed by an electrochemical dealloying process to selectively etch away Ag atoms. The surface properties of nanoporous membranes were investigated by energy-dispersive x-ray spectroscopy (EDS), scanning electron microscopy (SEM), atomic force microscopy (AFM), a documentation system, and a gel image system (Gel Doc Imager). A single strand of probe deoxyribonucleic acid (DNA) was immobilized onto the electrode surface by physical adsorption. The DNA probe and target hybridization were measured using a lock-in amplifier and an electrochemical impedance spectroscope (EIS). The nanoporous Pt-rich electrode-based DNA sensor offers a fast response time of 3.7 s, with a limit of detection (LOD) of 4.35 × 10-10 M of DNA target.

Effect of Membrane Tension on the Electric Field and Dipole Potential of Lipid Bilayer Membrane

PubMed Central

Warshaviak, Dora Toledo; Muellner, Michael J.; Chachisvilis, Mirianas

2011-01-01

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45 mV in the physiologically relevant range of membrane tension values (0 to 15 dyn/cm). The electrostatic field exhibits a peak (~0.8×109 V/m) near the water/lipid interface which shifts by 0.9 Å towards the bilayer center at 15 dyn/cm. Maximum membrane tension of 15 dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states. PMID:21722624

Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

PubMed

Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

2014-12-11

The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

Proteomic analysis of symbiosome membranes in Cnidaria-dinoflagellate endosymbiosis.

PubMed

Peng, Shao-En; Wang, Yu-Bao; Wang, Li-Hsueh; Chen, Wan-Nan Uang; Lu, Chi-Yu; Fang, Lee-Shing; Chen, Chii-Shiarng

2010-03-01

Symbiosomes are specific intracellular membrane-bound vacuoles containing microalgae in a mutualistic Cnidaria (host)-dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin-XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X-100 soluble and insoluble fractions, were subjected to 2-D SDS-PAGE and identified by MS using an LC-nano-ESI-MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti-apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.

Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe.

PubMed

Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie

2017-10-02

Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanoscale Probing of Electrical Signals in Biological Systems

DTIC Science & Technology

2012-03-18

Membranes Anodized aluminum oxide ( AAO ) is an ideal prototype substrate for studying ion transport through nanoporous membranes . For optimal...electrochemical microscopy, scanning ion conductance microscopy, nanoporous membranes , anodized aluminum oxide , atomic layer deposition, focused ion beam...capacity. This approach utilizes atomic layer deposition (ALD) of a thin conformal Ir film into a nanoporous anodized aluminum oxide (

Proton Transfer in Perfluorosulfonic Acid Fuel Cell Membranes with Differing Pendant Chains and Equivalent Weights.

PubMed

Thomaz, Joseph E; Lawler, Christian M; Fayer, Michael D

2017-05-04

Proton transfer in the nanoscopic water channels of polyelectrolyte fuel cell membranes was studied using a photoacid, 8-hydroxypyrene-1,3,6-trisulfonic acid sodium salt (HPTS), in the channels. The local environment of the probe was determined using 8-methoxypyrene-1,3,6-trisulfonic acid sodium salt (MPTS), which is not a photoacid. Three fully hydrated membranes, Nafion (DuPont) and two 3M membranes, were studied to determine the impact of different pendant chains and equivalent weights on proton transfer. Fluorescence anisotropy and excited state population decay data that characterize the local environment of the fluorescent probes and proton transfer dynamics were measured. The MPTS lifetime and anisotropy results show that most of the fluorescent probes have a bulk-like water environment with a relatively small fraction interacting with the channel wall. Measurements of the HPTS protonated and deprotonated fluorescent bands' population decays provided information on the proton transport dynamics. The decay of the protonated band from ∼0.5 ns to tens of nanoseconds is in part determined by dissociation and recombination with the HPTS, providing information on the ability of protons to move in the channels. The dissociation and recombination is manifested as a power law component in the protonated band fluorescence decay. The results show that equivalent weight differences between two 3M membranes resulted in a small difference in proton transfer. However, differences in pendant chain structure did significantly influence the proton transfer ability, with the 3M membranes displaying more facile transfer than Nafion.

Polyvinylidene fluoride membranes probed by electrochemical impedance spectroscopy

NASA Astrophysics Data System (ADS)

Luo, Qi-Zhao; Huang, Qing; Chen, Zhe; Yao, Lei; Fu, Ping; Lin, Zhi-Dong

2018-06-01

Electrochemical impedance spectroscopy (EIS) has been applied to characterize the structure of polyvinylidene fluoride (PVDF) membranes. The characteristic frequency, which directly obtained from the original EIS data, was used to clarify the difference of the membranes’ structures. The experimental data indicated the equivalence between the characteristic frequency and the membrane resistance fitted from the equivalent circuit. The results evidenced that the characteristic frequency obtained directly from original EIS data without any fitting calculation can be used for in situ characterizing a membrane instead of the membrane resistance.

Determining the Orientation and Localization of Membrane-Bound Peptides

PubMed Central

Hohlweg, Walter; Kosol, Simone; Zangger, Klaus

2012-01-01

Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance. PMID:22044140

A LOW-E MAGIC ANGLE SPINNING PROBE FOR BIOLOGICAL SOLID STATE NMR AT 750 MHz

PubMed Central

McNeill, Seth A.; Gor’kov, Peter L.; Shetty, Kiran; Brey, William W.; Long, Joanna R.

2009-01-01

Crossed-coil NMR probes are a useful tool for reducing sample heating for biological solid state NMR. In a crossed-coil probe, the higher frequency 1H field, which is the primary source of sample heating in conventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltage is required, the electric field across the sample and the resultant heating is reduced. In this work we describe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual resonator. This dual resonator approach, referred to as “Low-E,” was originally developed to reduce heating in samples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins in lipid bilayers, via MAS techniques requires large sample volumes at high field to obtain spectra with adequate signal-to-noise ratio under physiologically relevant conditions. With the Low-E approach, we are able to obtain homogeneous and sufficiently strong radiofrequency fields for both 1H and 13C frequencies in a 4 mm probe with a 1H frequency of 750 MHz. The performance of the probe using windowless dipolar recoupling sequences is demonstrated on model compounds as well as membrane embedded peptides. PMID:19138870

Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors*

PubMed Central

Balla, András; Tóth, Dániel J.; Soltész-Katona, Eszter; Szakadáti, Gyöngyi; Erdélyi, László Sándor; Várnai, Péter; Hunyady, László

2012-01-01

Initiation and termination of signaling of the type I angiotensin receptor (AT1-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT1-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT1-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT1-R upon AngII stimulus with those of [Sar1,Ile8]AngII or [Sar1,Ile4,Ile8]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT1-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments. PMID:22291018

A homogeneous and "off-on" fluorescence aptamer-based assay for chloramphenicol using vesicle quantum dot-gold colloid composite probes.

PubMed

Miao, Yang-Bao; Ren, Hong-Xia; Gan, Ning; Zhou, You; Cao, Yuting; Li, Tianhua; Chen, Yinji

2016-07-27

In this work, a novel homogeneous and signal "off-on" aptamer based fluorescence assay was successfully developed to detect chloramphenicol (CAP) residues in food based on the fluorescence resonance energy transfer (FRET). The vesicle nanotracer was prepared through labeling single stranded DNA binding protein (SSB) on limposome-CdSe/ZnS quantum dot (SSB/L-QD) complexes. It was worth mentioning that the signal tracer (SSB/L-QD) with vesicle shape, which was fabricated being encapsulated with a number of quantum dots and SSB. The nanotracer has excellent signal amplification effects. The vesicle composite probe was formed by combining aptamer labeled nano-gold (Au-Apt) and SSB/L-QD. Which based on SSB's specific affinity towards aptamer. This probe can't emit fluoresce which is in "off" state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor. When CAP was added in the composite probe solution, the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe, which can turn the "off" signal of SSB/L-QD tracer into "on" state. The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM. The vesicle probes with size of 88 nm have strong signal amplification. Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane. Besides, it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA, verifying its accuracy and reliability. Copyright © 2016 Elsevier B.V. All rights reserved.

Concentration of isoprene in artificial and thylakoid membranes.

PubMed

Harvey, Christopher M; Li, Ziru; Tjellström, Henrik; Blanchard, Gary J; Sharkey, Thomas D

2015-10-01

Isoprene emission protects plants from a variety of abiotic stresses. It has been hypothesized to do so by partitioning into cellular membranes, particularly the thylakoid membrane. At sufficiently high concentrations, this partitioning may alter the physical properties of membranes. As much as several per cent of carbon taken up in photosynthesis is re-emitted as isoprene but the concentration of isoprene in the thylakoid membrane of rapidly emitting plants has seldom been considered. In this study, the intramembrane concentration of isoprene in phosphatidylcholine liposomes equilibrated to a physiologically relevant gas phase concentration of 20 μL L(-1) isoprene was less than predicted by ab initio calculations based on the octanol-water partitioning coefficient of isoprene while the concentration in thylakoid membranes was more. However, the concentration in both systems was roughly two orders of magnitude lower than previously assumed. High concentrations of isoprene (2000 μL L(-1) gas phase) failed to alter the viscosity of phosphatidylcholine liposomes as measured with perylene, a molecular probe of membrane structure. These results strongly suggest that the physiological concentration of isoprene within the leaves of highly emitting plants is too low to affect the dynamics of thylakoid membrane acyl lipids. It is speculated that isoprene may bind to and modulate the dynamics of thylakoid embedded proteins.

Development of Nano/Micro Probes for Femtoliter Volume and Single Cell Measurements

NASA Astrophysics Data System (ADS)

Gao, Yang

Single cell analysis has recently emerged as an important field of biomedical re- search. It is now clear that heterogeneity of cell metabolism functions in complex biological systems is correlated to changes in biological function and disease processes. A variety of nano/micro probes were developed to enable investigation of cells properties such as membrane stiffness, pH value. However, very few designs were focused on single cell metabolic function studies. There is a critical need for technologies that provide analysis of heterogeneity of cell metabolic functions, especially on metabolism. Nevertheless, the few existing approaches suffer from fundamental defects and need to be improved. This work focused on developing nano/micro probes that are suitable for single cell functionality investigation. Both types of probes are designed to measure cell-to-cell/time-to-time heterogeneity in metabolic functions over a long period of time. Lab-made carbon nanoprobes were developed especially for electro-physiological measurement. The unique structure of the carbon nanoprobes makes them suitable for important intracellular applications like trans-membrane potential measurements and various electrochemical measurement for cell function studies. While it is important of have ability to carry out intracellular measure, there are also occasions where the information of a cell as a whole is collected. One of the most important indicator of a cells metabolic functions is cell respiration rate/oxygen consumption rate. A micro-perfusion based multi-functional single cell sensing probe was the developed to carry out measurements on cell as a whole. Formed by a double-barrel theta pipette, the perfusion flow enables the direct measurement of the metabolic flux for example oxygen consumption rate. In conclusion, this work developed nano/micro-probes as novel single cell investigation tools. The data acquired from these tools could provide valuable assistance on applications including cell metabolism studies, cancer diagnoses, and therapy evaluations.

Electrochemical Impedance Spectroscopy for Real-Time Detection of Lipid Membrane Damage Based on a Porous Self-Assembly Monolayer Support.

PubMed

Zhang, Meng; Zhai, Qingyu; Wan, Liping; Chen, Li; Peng, Yu; Deng, Chunyan; Xiang, Juan; Yan, Jiawei

2018-06-19

Layer-by-layer dissolution and permeable pore formation are two typical membrane damage pathways, which induce membrane function disorder and result in serious disease, such as Alzheimer's disease, Keshan disease, Sickle-cell disease, and so on. To effectively distinguish and sensitively monitor these two typical membrane damage pathways, a facile electrochemical impedance strategy was developed on a porous self-assembly monolayer (pSAM) supported bilayer lipid membrane (BLM). The pSAM was prepared by selectively electrochemical reductive desorption of the mercaptopropionic acid in a mixed mercaptopropionic acid/11-mercaptoundecanoic acid self-assembled monolayer, which created plenty of nanopores with tens of nanometers in diameter and several nanometers in height (defined as inner-pores). The ultralow aspect ratio of the inner-pores was advantageous to the mass transfer of electrochemical probe [Fe(CN) 6 ] 3-/4- , simplifying the equivalent electric circuit for electrochemical impedance spectroscopy analysis at the electrode/membrane interface. [Fe(CN) 6 ] 3-/4- transferring from the bulk solution into the inner-pore induce significant changes of the interfacial impedance properties, improving the detection sensitivity. Based on these, the different membrane damage pathways were effectively distinguished and sensitively monitored with the normalized resistance-capacitance changes of inner-pore-related parameters including the electrolyte resistance within the pore length ( R pore ) and the metal/inner-pore interfacial capacitance ( C pore ) and the charge-transfer resistance ( R ct-in ) at the metal/inner-pore interface.

Direct Prediction of EPR Spectra from Lipid Bilayers: Understanding Structure and Dynamics in Biological Membranes.

PubMed

Catte, Andrea; White, Gaye F; Wilson, Mark R; Oganesyan, Vasily S

2018-06-02

Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer. Our results demonstrate very good agreement with experiment and thus confirm the accuracy of the latest lipid force fields. The atomic resolution of the simulations allows the interpretation of the molecular motions and interactions in terms of their impact on the sensitive EPR line shapes. Direct versus indirect effects of cholesterol on the dynamics of spin probes are analysed. Given the complexity of structural organisation in lipid bilayers, the advantage of using a combined MD-EPR simulation approach is two-fold. Firstly, prediction of EPR line shapes directly from MD trajectories of actual phospholipid structures allows unambiguous interpretation of EPR spectra of biological membranes in terms of complex motions. Secondly, such an approach provides an ultimate test bed for the up-to-date MD simulation models employed in the studies of biological membranes, an area that currently attracts great attention. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gaseous Microemboli and the Influence of Microporous Membrane Oxygenators

PubMed Central

Weitkemper, Heinz-H.; Oppermann, Bernd; Spilker, Andreas; Knobl, Hermann-J.; Körfer, Reiner

2005-01-01

Abstract: Gaseous microemboli (GME) are still an unsolved problem of extracorporeal circuits. They are associated with organ injury during cardiopulmonary bypass. Microbubbles of different sizes and number are generated in the blood as the result of different components of the extracorporeal circuit as well as surgical maneuvers. The aim of our study was to observe the behavior of microporous membrane oxygenators to GME in the daily use and in an in vitro model. For the detection of microbubbles, we used a two-channel ultrasonic bubble counter based on 2-MHz Doppler-System with special ultrasound probes. The amount and size of GME were monitored before and after membrane. In 28 scheduled cases with 3 different oxygenators and variability of surgical procedures, we observed the bubble activity in the extracorporeal circuit. In addition, we used an in-vitro model to study the ability of six different oxygenators by removing air in various tests. The oxygenators tested were manufactured with different membrane technologies. The results of our investigations showed varying membrane design lead to a partial removal of GME as well as a change in size and numbers of microbubbles. PMID:16350377

Bacterial cellulose membrane as separation medium

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

Shibazaki, Hideki; Kuga, Shigenori; Onabe, Fumihiko

1993-11-10

A thin membrane of bacterial cellulose (BC) obtained from Acetobacter culture was tested for its performance as a dialysis membrane in aqueous systems. The BC membrane showed superior mechanical strength to that of a dialysis-grade regenerated cellulose membrane, allowing the use of a thinner membrane than the latter. As a result, the BC membrane gave higher permeation rates for poly(ethylene glycols) as probe solutes. The cutoff molecular weight of the original BC membrane, significantly greater than that of regenerated cellulose, could be modified by concentrated alkali treatments of the membrane. The nature of the change at the ultrastructural level causedmore » by the alkali treatments was studied by X-ray diffraction and scanning electron microscopy.« less

Watching individual molecules flex within lipid membranes using SERS

NASA Astrophysics Data System (ADS)

Taylor, Richard W.; Benz, Felix; Sigle, Daniel O.; Bowman, Richard W.; Bao, Peng; Roth, Johannes S.; Heath, George R.; Evans, Stephen D.; Baumberg, Jeremy J.

2014-08-01

Interrogating individual molecules within bio-membranes is key to deepening our understanding of biological processes essential for life. Using Raman spectroscopy to map molecular vibrations is ideal to non-destructively `fingerprint' biomolecules for dynamic information on their molecular structure, composition and conformation. Such tag-free tracking of molecules within lipid bio-membranes can directly connect structure and function. In this paper, stable co-assembly with gold nano-components in a `nanoparticle-on-mirror' geometry strongly enhances the local optical field and reduces the volume probed to a few nm3, enabling repeated measurements for many tens of minutes on the same molecules. The intense gap plasmons are assembled around model bio-membranes providing molecular identification of the diffusing lipids. Our experiments clearly evidence measurement of individual lipids flexing through telltale rapid correlated vibrational shifts and intensity fluctuations in the Raman spectrum. These track molecules that undergo bending and conformational changes within the probe volume, through their interactions with the environment. This technique allows for in situ high-speed single-molecule investigations of the molecules embedded within lipid bio-membranes. It thus offers a new way to investigate the hidden dynamics of cell membranes important to a myriad of life processes.

On the mechanics of growing thin biological membranes

NASA Astrophysics Data System (ADS)

Rausch, Manuel K.; Kuhl, Ellen

2014-02-01

Despite their seemingly delicate appearance, thin biological membranes fulfill various crucial roles in the human body and can sustain substantial mechanical loads. Unlike engineering structures, biological membranes are able to grow and adapt to changes in their mechanical environment. Finite element modeling of biological growth holds the potential to better understand the interplay of membrane form and function and to reliably predict the effects of disease or medical intervention. However, standard continuum elements typically fail to represent thin biological membranes efficiently, accurately, and robustly. Moreover, continuum models are typically cumbersome to generate from surface-based medical imaging data. Here we propose a computational model for finite membrane growth using a classical midsurface representation compatible with standard shell elements. By assuming elastic incompressibility and membrane-only growth, the model a priori satisfies the zero-normal stress condition. To demonstrate its modular nature, we implement the membrane growth model into the general-purpose non-linear finite element package Abaqus/Standard using the concept of user subroutines. To probe efficiently and robustness, we simulate selected benchmark examples of growing biological membranes under different loading conditions. To demonstrate the clinical potential, we simulate the functional adaptation of a heart valve leaflet in ischemic cardiomyopathy. We believe that our novel approach will be widely applicable to simulate the adaptive chronic growth of thin biological structures including skin membranes, mucous membranes, fetal membranes, tympanic membranes, corneoscleral membranes, and heart valve membranes. Ultimately, our model can be used to identify diseased states, predict disease evolution, and guide the design of interventional or pharmaceutic therapies to arrest or revert disease progression.

On the mechanics of growing thin biological membranes

PubMed Central

Rausch, Manuel K.; Kuhl, Ellen

2013-01-01

Despite their seemingly delicate appearance, thin biological membranes fulfill various crucial roles in the human body and can sustain substantial mechanical loads. Unlike engineering structures, biological membranes are able to grow and adapt to changes in their mechanical environment. Finite element modeling of biological growth holds the potential to better understand the interplay of membrane form and function and to reliably predict the effects of disease or medical intervention. However, standard continuum elements typically fail to represent thin biological membranes efficiently, accurately, and robustly. Moreover, continuum models are typically cumbersome to generate from surface-based medical imaging data. Here we propose a computational model for finite membrane growth using a classical midsurface representation compatible with standard shell elements. By assuming elastic incompressibility and membrane-only growth, the model a priori satisfies the zero-normal stress condition. To demonstrate its modular nature, we implement the membrane growth model into the general-purpose non-linear finite element package Abaqus/Standard using the concept of user subroutines. To probe efficiently and robustness, we simulate selected benchmark examples of growing biological membranes under different loading conditions. To demonstrate the clinical potential, we simulate the functional adaptation of a heart valve leaflet in ischemic cardiomyopathy. We believe that our novel approach will be widely applicable to simulate the adaptive chronic growth of thin biological structures including skin membranes, mucous membranes, fetal membranes, tympanic membranes, corneoscleral membranes, and heart valve membranes. Ultimately, our model can be used to identify diseased states, predict disease evolution, and guide the design of interventional or pharmaceutic therapies to arrest or revert disease progression. PMID:24563551

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Physical consequences of the mitochondrial targeting of single-walled carbon nanotubes probed computationally

NASA Astrophysics Data System (ADS)

Chistyakov, V. A.; Zolotukhin, P. V.; Prazdnova, E. V.; Alperovich, I.; Soldatov, A. V.

2015-06-01

Experiments by F. Zhou and coworkers (2010) [16] showed that mitochondria are the main target of the cellular accumulation of single-walled carbon nanotubes (SWCNTs). Our in silico experiments, based on geometrical optimization of the system consisting of SWCNT+proton within Density Functional Theory, revealed that protons can bind to the outer side of SWCNT so generating a positive charge. Calculation results allow one to propose the following mechanism of SWCNTs mitochondrial targeting. SWCNTs enter the space between inner and outer membranes of mitochondria, where the excess of protons has been formed by diffusion. In this compartment SWCNTs are loaded with protons and acquire positive charges distributed over their surface. Protonation of hydrophobic SWCNTs can also be carried out within the mitochondrial membrane through interaction with the protonated ubiquinone. Such "charge loaded" particles can be transferred as "Sculachev ions" through the inner membrane of the mitochondria due to the potential difference generated by the inner membrane. Physiological consequences of the described mechanism are discussed.

Structural design of intrinsically fluorescent oxysterols.

PubMed

Nåbo, Lina J; Modzel, Maciej; Krishnan, Kathiresan; Covey, Douglas F; Fujiwara, Hideji; Ory, Daniel S; Szomek, Maria; Khandelia, Himanshu; Wüstner, Daniel; Kongsted, Jacob

2018-05-01

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

Asymmetry of plasma membrane lipid order in Madin-Darby Canine Kidney cells.

PubMed

Le Grimellec, C; Friedlander, G; Giocondi, M C

1988-07-01

Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the plasma membrane of polarized Madin-Darby Canine Kidney cells (MDCK) grown on glass cover slips and labeled by 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific marker of the plasma membrane of living cells. Fluorescence microscopy, back-exchange, and quenching experiments indicated that TMA-DPH labeled the highly ordered (r greater than or equal to 0.32, 37 degrees C) apical domain of the plasma membrane of confluent monolayers. Opening of tight junctions or addition of the probe to cell suspensions resulted in a homogeneous distribution of TMA-DPH over the cell surface and in a marked decrease in anisotropy (0.27 less than or equal to r less than or equal to 0.29) that was due neither to a direct effect of Ca2+ on the probe nor to a change in fluorescence lifetime. Our data indicate that the apical domain, likely the external leaflet, of the plasma membrane of polarized MDCK cells is much more ordered than its basolateral counterpart.

A membrane filtering method for the purification of giant unilamellar vesicles.

PubMed

Tamba, Yukihiro; Terashima, Hiroaki; Yamazaki, Masahito

2011-07-01

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10-30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Bioorthogonal probes for imaging sterols in cells.

PubMed

Jao, Cindy Y; Nedelcu, Daniel; Lopez, Lyle V; Samarakoon, Thilani N; Welti, Ruth; Salic, Adrian

2015-03-02

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative validation of an air-coupled ultrasonic probe model by Interferometric laser tomography

NASA Astrophysics Data System (ADS)

Revel, G. M.; Pandarese, G.; Cavuto, A.

2012-06-01

The present paper describes the quantitative validation of a finite element (FE) model of the ultrasound beam generated by an air coupled non-contact ultrasound transducer. The model boundary conditions are given by vibration velocities measured by laser vibrometry on the probe membrane. The proposed validation method is based on the comparison between the simulated 3D pressure field and the pressure data measured with interferometric laser tomography technique. The model details and the experimental techniques are described in paper. The analysis of results shows the effectiveness of the proposed approach and the possibility to quantitatively assess and predict the generated acoustic pressure field, with maximum discrepancies in the order of 20% due to uncertainty effects. This step is important for determining in complex problems the real applicability of air-coupled probes and for the simulation of the whole inspection procedure, also when the component is designed, so as to virtually verify its inspectability.

Discrimination of three types of homopolymers in single-stranded DNA with solid-state nanopores through external control of the DNA motion.

PubMed

Akahori, Rena; Yanagi, Itaru; Goto, Yusuke; Harada, Kunio; Yokoi, Takahide; Takeda, Ken-Ichi

2017-08-22

To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane. We demonstrated that a single-stranded DNA could be inserted into and removed from a nanopore in our experimental system. The number of different ionic-current levels observed while DNA remained in the nanopore corresponded to the number of different types of homopolymers in the DNA.

Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition

PubMed Central

Brignac-Huber, Lauren M.; Reed, James R.; Eyer, Marilyn K.

2013-01-01

Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (lo) domains. The goal of this study was to determine if lo domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in “ER-like” lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form lo domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model lo-forming systems demonstrated the coexistence of lo- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the lo regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into lo membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing lo domains. PMID:23963955

Fluidity of pea root plasma membranes under altered gravity

NASA Astrophysics Data System (ADS)

Klymchuk, D. O.; Baranenko, V. V.; Vorobyova, T. V.; Dubovoy, V. D.

This investigation aims to determine whether clinorotation 2 rev min of pea Pisum sativum L seedlings induces the alterations in the physical-chemical properties of cellular membranes including the plasma membrane fluidity The last is an important regulator of functional activity of membrane enzymes The plasma membranes were isolated by aqueous two-phase partitioning from roots of 6-day old pea seedlings The membrane fluidity was examined by fluorescence spectroscopy using pyrene probe The plasma membrane vesicles with known protein concentration were added to the incubation buffer to a final concentration of 50 mu g of protein per ml A small amount by 1 mu l of pyrene solution in 2-propanol was added to the incubation mixture to a final probe concentration 5 mu M at constant mixing Fluorescence spectra were measured using a Perkin-Elmer LS-50 spectrofluorometer Perkin-Elmer England Pyrene was excited at 337 nm and fluorescence intensity of monomers I M and excimers I E were measured at 393 and 470 nm respectively The I E I M ratios were 0 081 pm 0 003 and 0 072 pm 0 004 in preparations obtained from clinorotated and the control seedlings respectively This fact indicates that rotation on the clinostat increases the membrane fluidity Compared with controls clinorotated seedlings have also showed a reduced growth and a higher level of total unsaturated fatty acids determined by gas chromatography The factors that influence on the fluidity of membrane lipids in bilayer appear to be the

Trans-cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells.

PubMed

Chmyrov, Volodymyr; Spielmann, Thiemo; Hevekerl, Heike; Widengren, Jerker

2015-06-02

Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

Southern blotting.

PubMed

Brown, T

2001-05-01

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.

CO2 flux monitoring using Continuous Timeseries-Forced Diffusion (CT-FD): Development, Validation

NASA Astrophysics Data System (ADS)

McArthur, G. S.; Risk, D. A.; Nickerson, N. R.; Creelman, C. A.; Beltrami, H.

2009-12-01

Land-based CO2 flux measurements are a key indicator of the biological, chemical and physical processes occurring in the soil. While highly dense temporal flux measurements can be acquired using Eddy Covariance towers, or flux chambers, the challenge of gathering data that is rich both temporally and spatially persists. Over the past two years we have developed a new technique for measuring soil CO2 fluxes, called continuous timeseries-forced diffusion (CT-FD) attempts to satisfy the need for spatially and temporally rich data. The CT-FD probe consists of a Vaisala CO2 sensor, embodied in a PVC casing, with tear/UV resistant Tyvek membranes at both the inlet and outlet. The probe delivers continuous flux data and can be inexpensively replicated across the landscape.The CT-FD technique works by forcing a known diffusive regime between the soil and the atmosphere, allowing the calculation of fluxes across the soil/atmosphere boundary to be made from; the internal concentration of a CT-FD probe placed at the soil surface; and a common reference probe designed to capture the atmospheric CO2. For every concentration measurement, the difference between the probe and the reference concentration is indicative of a unique flux value. Here we examine properties of the instrument and method, as documented by a long series of developmental studies involving numerical gas transport modeling, laboratory and field experiments. A suite of 1D and 3D modeling experiments were needed to optimize embodiment and geometries of the probe. These show that the probe should have a relatively long collar, with relatively high diffusivity made possible by having large, highly diffusive membranes, both of which help to induce 1D movement of gases into the probe and reduce the lateral diffusion around the probe. Modeling also shows that correction for lateral diffusion is feasible. As for error, sensor error transfers linearly to errors in the flux, and that the sensor can be used in non free-atmospheric environments, for example when snow falls and persists. For calibration purposes we designed and built a flux generator, allowing us to test different mathematical approaches for reliability and calibrations which is done by plotting the known flux against the difference between probe and atmospheric CO2 measurements. Validation of the technique was also carried out in the lab using soil plots in which heating cables drove diurnal microbial CO2 production, and we found CT-FD to have an excellent correspondence with LI-8100, showing similar accuracy and precision. Using CT-FD we performed two extensive winter campaigns and one summer campaign in a salt marsh with both CO2-capable and CH4-capable (METS sensor-based) probes. Here we found the CT-FD capable of long, unattended deployments, continued effectiveness when buried under deep snowpack, exposed to long term freezing temperatures, and heavy rain events.

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

DOE PAGES

Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; ...

2015-07-24

We report that design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) andmore » second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Lastly, our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.« less

Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560

Noninvasive probes of mitochondrial molecular motors

NASA Astrophysics Data System (ADS)

Nawarathna, Dharmakeerthna; Claycomb, James

2005-03-01

We report on a noninvasive method of probing mitochondrial molecular motors using nonlinear dielectric spectroscopy. It has been found previously that enzymes in the plasma membrane, particularly H+ ATPase, result in a strong low frequency (less than 100 Hz) nonlinear harmonic response. In this study, we find evidence that molecular motors located in the inner membranes of mitochondria cause the generation of harmonics at relatively high frequencies (1 - 30 kHz). In particular, we find that potassium cyanide (KCN), a respiratory inhibitor that binds to cytochrome c oxidase and thus prevents transport of protons across the mitochondrial inner membrane, suppresses the harmonic response. We observe this behavior in yeast (S. cerevisiae), a eucaryote that typically contains about 300 mitochondria, and B. indicas, a procaryote believed to be related to the ancient ancestor of mitochondria. Our current modeling efforts are focusing on a Brownian ratchet model of the F0 unit of ATP synthase, a remarkable molecular turbine driven by the proton gradient across the mitochondrial inner membrane.

A miniaturized laser-Doppler-system in the ear canal

NASA Astrophysics Data System (ADS)

Schmidt, T.; Gerhardt, U.; Kupper, C.; Manske, E.; Witte, H.

2013-03-01

Gathering vibrational data from the human middle ear is quite difficult. To this date the well-known acoustic probe is used to estimate audiometric parameters, e.g. otoacoustic emissions, wideband reflectance and the measurement of the stapedius reflex. An acoustic probe contains at least one microphone and one loudspeaker. The acoustic parameter determination of the ear canal is essential for the comparability of test-retest measurement situations. Compared to acoustic tubes, the ear canal wall cannot be described as a sound hard boundary. Sound energy is partly absorbed by the ear canal wall. In addition the ear canal features a complex geometric shape (Stinson and Lawton1). Those conditions are one reason for the inter individual variability in input impedance measurement data of the tympanic membrane. The method of Laser-Doppler-Vibrometry is well described in literature. Using this method, the surface velocity of vibrating bodies can be determined contact-free. Conventional Laser-Doppler-Systems (LDS) for auditory research are mounted on a surgical microscope. Assuming a free line of view to the ear drum, the handling of those laser-systems is complicated. We introduce the concept of a miniaturized vibrometer which is supposed to be applied directly in the ear canal for contact-free measurement of the tympanic membrane surface vibration. The proposed interferometer is based on a Fabry-Perot etalon with a DFB laser diode as light source. The fiber-based Fabry-Perot-interferometer is characterized by a reduced size, compared to e.g. Michelson-, or Mach-Zehnder-Systems. For the determination of the phase difference in the interferometer, a phase generated carrier was used. To fit the sensor head in the ear canal, the required shape of the probe was generated by means of the geometrical data of 70 ear molds. The suggested prototype is built up by a singlemode optical fiber with a GRIN-lens, acting as a fiber collimator. The probe has a diameter of 1.8 mm and a length of 5 mm.

Scanning transmission X-ray microscopy probe for in situ mechanism study of graphene-oxide-based resistive random access memory.

PubMed

Nho, Hyun Woo; Kim, Jong Yun; Wang, Jian; Shin, Hyun-Joon; Choi, Sung-Yool; Yoon, Tae Hyun

2014-01-01

Here, an in situ probe for scanning transmission X-ray microscopy (STXM) has been developed and applied to the study of the bipolar resistive switching (BRS) mechanism in an Al/graphene oxide (GO)/Al resistive random access memory (RRAM) device. To perform in situ STXM studies at the C K- and O K-edges, both the RRAM junctions and the I0 junction were fabricated on a single Si3N4 membrane to obtain local XANES spectra at these absorption edges with more delicate I0 normalization. Using this probe combined with the synchrotron-based STXM technique, it was possible to observe unique chemical changes involved in the BRS process of the Al/GO/Al RRAM device. Reversible oxidation and reduction of GO induced by the externally applied bias voltages were observed at the O K-edge XANES feature located at 538.2 eV, which strongly supported the oxygen ion drift model that was recently proposed from ex situ transmission electron microscope studies.

A chromogenic and fluorogenic rhodol-based chemosensor for hydrazine detection and its application in live cell bioimaging.

PubMed

Tiensomjitr, Khomsan; Noorat, Rattha; Chomngam, Sinchai; Wechakorn, Kanokorn; Prabpai, Samran; Kanjanasirirat, Phongthon; Pewkliang, Yongyut; Borwornpinyo, Suparerk; Kongsaeree, Palangpon

2018-04-15

A rhodol-based fluorescent probe has been developed as a selective hydrazine chemosensor using levulinate as a recognition site. The rhodol levulinate probe (RL) demonstrated high selectivity and sensitivity toward hydrazine among other molecules. The chromogenic response of RL solution to hydrazine from colorless to pink could be readily observed by the naked eye, while strong fluorescence emission could be monitored upon excitation at 525 nm. The detection process occurred via a ring-opening process of the spirolactone initiated by hydrazinolysis, triggering the fluorescence emission with a 53-fold enhancement. The probe rapidly reacted with hydrazine in aqueous medium with the detection limit of 26 nM (0.83 ppb), lower than the threshold limit value (TLV) of 10 ppb suggested by the U.S. Environmental Protection Agency. Furthermore, RL-impregnated paper strips could detect hydrazine vapor. For biological applicability of RL, its membrane-permeable property led to bioimaging of hydrazine in live HepG2 cells by confocal fluorescence microscopy. Copyright © 2018 Elsevier B.V. All rights reserved.

Non-invasive optoacoustic probing of the density and stiffness of single biological cells

NASA Astrophysics Data System (ADS)

Dehoux, T.; Audoin, B.

2012-12-01

Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accurately for a reference fluid. Similar analysis performed in a single vegetal cell also suggests high sensitivity to the state of the transducer-cell interface, and notably to the presence of the plasma membrane that encloses the cell vacuole.

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

PubMed Central

Dyszy, Fábio; Pinto, Andressa P. A.; Araújo, Ana P. U.; Costa-Filho, Antonio J.

2013-01-01

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs. PMID:23555925

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

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

Gaibelet, Gérald; CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette; Tercé, François

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins couldmore » be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD-Chol non-specifically delivered to the cells.« less

Intrinsic property measurement of surfactant-templated mesoporous silica films using time-resolved single-molecule imaging.

PubMed

Kennard, Raymond; DeSisto, William J; Giririjan, Thanu Praba; Mason, Michael D

2008-04-07

Mesoporous silica membranes fabricated by the surfactant-templated sol-gel process have received attention because of the potential to prepare membranes with a narrow pore size distribution and ordering of the interconnected pores. Potential applications include ultrafiltration, biological separations and drug delivery, and separators in lithium-ion batteries. Despite advancements in synthesis and characterization of these membranes, a quantitative description of the membrane microstructure remains a challenge. Currently the membrane microstructure is characterized by the combination of results from several techniques, i.e., gas permeance testing, x-ray diffraction scanning electron microscopy, transmission electron microscopy, and permporometry. The results from these ensemble methods are then compiled and the data fitted to a particular flow model. Although these methods are very effective in determining membrane performance, general pore size distribution, and defect concentration, they are unable to monitor molecular paths through the membrane and quantitatively measure molecular interactions between the molecular specie and pore network. Single-molecule imaging techniques enable optical measurements that probe materials on nanometer length scales through observation of individual molecules without the influence of averaging. Using single-molecule imaging spectroscopy, we can quantitatively characterize the interaction between the probe molecule and the interior of the pore within mesoporous silica membranes. This approach is radically different from typical membrane characterization methods in that it has the potential to spatially sample the underlying pore structure distribution, the surface energy, and the transport properties. Our hope is that this new fundamental knowledge can be quantitatively linked to both the preparation and the performance of membranes, leading to the advancement of membrane science and technology. Fluorescent molecules, 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5 nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50 degrees C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.

Intrinsic property measurement of surfactant-templated mesoporous silica films using time-resolved single-molecule imaging

NASA Astrophysics Data System (ADS)

Kennard, Raymond; DeSisto, William J.; Giririjan, Thanu Praba; Mason, Michael D.

2008-04-01

Mesoporous silica membranes fabricated by the surfactant-templated sol-gel process have received attention because of the potential to prepare membranes with a narrow pore size distribution and ordering of the interconnected pores. Potential applications include ultrafiltration, biological separations and drug delivery, and separators in lithium-ion batteries. Despite advancements in synthesis and characterization of these membranes, a quantitative description of the membrane microstructure remains a challenge. Currently the membrane microstructure is characterized by the combination of results from several techniques, i.e., gas permeance testing, x-ray diffraction scanning electron microscopy, transmission electron microscopy, and permporometry. The results from these ensemble methods are then compiled and the data fitted to a particular flow model. Although these methods are very effective in determining membrane performance, general pore size distribution, and defect concentration, they are unable to monitor molecular paths through the membrane and quantitatively measure molecular interactions between the molecular specie and pore network. Single-molecule imaging techniques enable optical measurements that probe materials on nanometer length scales through observation of individual molecules without the influence of averaging. Using single-molecule imaging spectroscopy, we can quantitatively characterize the interaction between the probe molecule and the interior of the pore within mesoporous silica membranes. This approach is radically different from typical membrane characterization methods in that it has the potential to spatially sample the underlying pore structure distribution, the surface energy, and the transport properties. Our hope is that this new fundamental knowledge can be quantitatively linked to both the preparation and the performance of membranes, leading to the advancement of membrane science and technology. Fluorescent molecules, 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50°C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.

Lateral Distribution of NBD-PC Fluorescent Lipid Analogs in Membranes Probed by Molecular Dynamics-Assisted Analysis of Förster Resonance Energy Transfer (FRET) and Fluorescence Quenching

PubMed Central

Loura, Luís M. S.

2012-01-01

Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed. PMID:23203080

Lateral distribution of NBD-PC fluorescent lipid analogs in membranes probed by molecular dynamics-assisted analysis of Förster Resonance Energy Transfer (FRET) and fluorescence quenching.

PubMed

Loura, Luís M S

2012-11-08

Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed.

Visual detection of cyanide ions by membrane-based nanozyme assay.

PubMed

Lien, Chia-Wen; Unnikrishnan, Binesh; Harroun, Scott G; Wang, Chih-Min; Chang, Jia-Yaw; Chang, Huan-Tsung; Huang, Chih-Ching

2018-04-15

In this paper, we report a simple one-step synthesis of well-dispersed amorphous cobalt hydroxide/oxide-modified graphene oxide (CoO x H-GO) possessing peroxidase-like catalytic activity, and its application for the detection of H 2 O 2 , glucose, and CN - ions. CoO x H is formed and deposited in situ on the GO surface through the reaction between GO (size ~ 240nm) and Co 2+ in basic solution at room temperature. We investigated the enzyme-mimicking activity of the CoO x H-GO nanohybrid in detail via the H 2 O 2 -mediated oxidation of Amplex Red (AR) to form fluorescent resorufin. The peroxidase-like activity of CoO x H-GO is utilized herein for the quantitation of H 2 O 2 in a wide concentration range, from 100nM to 100μM. When coupled with glucose oxidase (GOD), the AR/CoO x H-GO system can determine glucose level in blood samples. Interestingly, cyanide ions (CN - ) significantly inhibit the catalytic activity of the CoO x H-GO nanohybrid, which allows for the construction of a probe for the detection of CN - in water samples and laboratory wastes. We fabricated a membrane-based CoO x H-GO probe for the visual detection of CN - by preparing a thin film of CoO x H-GO on a positively charged and porous nylon membrane (N + M). The CoO x H-GO/N + M operates on the principle that CN - inhibits the catalytic activity of CoO x H-GO towards the H 2 O 2 -mediated oxidation of AR to form reddish resorufin on the membrane. The intensity of the red color of the membrane decreases with increasing CN - concentration, which can be easily observed with the naked eye at the nanomolar level. This cost-effective sensing system allows for the rapid and simple determination of the concentrations of CN - in complicated wastewater samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules.

PubMed Central

Balcom, B J; Petersen, N O

1993-01-01

We have systematically investigated the probe size and shape dependence of lateral diffusion in model dimyristoyl phosphatidylcholine membranes. Linear hydrophobic polymers, which differ in length by an order of magnitude, were used to explore the effect on the lateral diffusion coefficient of hydrodynamic restrictions in the bilayer interior. The polymers employed are isoprenoid alcohols--citronellol, solanesol, and dolichol. Tracer lateral diffusion coefficients were measured by fluorescence photobleaching recovery. Despite the large difference in lengths, the nitrobenzoxadiazole labelled alcohols all diffuse at the rate of lipid self-diffusion (5.0 x 10(-12) m2 s-1, 29 degrees C) in the liquid crystal phase. Companion measurements in isotropic polymer solution, in gel phase lipid membranes and with nonpolar fluorescent polyaromatic hydrocarbons, show a marked dependence of the lateral diffusion coefficient on the probe molecule size. Our results in the liquid crystal phase are in accord with free area theory which asserts that lateral diffusion in the membrane is restricted by the surface-free area. Probe molecules which are significantly longer than the host phospholipid, seven times longer in the case of dolichol, are still restricted in their lateral motion by the surface properties of the bilayer in the liquid crystal phase. Fluorescence quenching experiments indicate that the nitrobenzoxadiazole label does not reside at the aqueous interface, although it must reside in close proximity according to the diffusion measurements. PMID:8218892

Mechanics of membrane-cytoskeleton attachment in Paramecium

NASA Astrophysics Data System (ADS)

Campillo, C.; Jerber, J.; Fisch, C.; Simoes-Betbeder, M.; Dupuis-Williams, P.; Nassoy, P.; Sykes, C.

2012-12-01

In this paper we assess the role of the protein MKS1 (Meckel syndrome type 1) in the cortical membrane mechanics of the ciliated protist Paramecium. This protein is known to be crucial in the process of cilium formation, and we investigate its putative role in membrane-cytoskeleton attachment. Therefore, we compare cells where the gene coding for MKS1 is silenced to wild-type cells. We found that scanning electron microscopy observation of the cell surface reveals a cup-like structure in wild-type cells that is lost in silenced cells. Since this structure is based on the underlying cytoskeleton, one hypothesis to explain this observation is a disruption of membrane attachment to the cytoskeleton in the absence of MKS1 that should affect plasma membrane mechanics. We test this by probing the mechanics of wild-type and silenced cells by micropipette aspiration. Strikingly, we observe that, at the same aspiration pressure, the membrane of silenced cells is easily aspirated by the micropipette whereas that of wild-type cells enters only at a moderate velocity, an effect that suggests a detachment of the membrane from the underlying cytoskeleton in silenced cells. We quantify this detachment by measuring the deformation of the cell cortex and the rate of cell membrane entry in the micropipette. This study offers a new perspective for the characterization of membrane-cytoskeleton attachment in protists and paves the way for a better understanding of the role of membrane-cortex attachment in cilium formation.

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

PubMed

Klotzsch, Enrico; Stiegler, Johannes; Ben-Ishay, Eldad; Gaus, Katharina

2015-04-01

Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor-ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling. Copyright © 2014 Elsevier B.V. All rights reserved.

A resumable two-photon fluorescent probe for Cu2+ and S2- based on magnetic silica core-shell Fe3O4@SiO2 nanoparticles and its application in bioimaging.

PubMed

Jiang, Huie; Liu, Yan; Luo, Weifang; Wang, Yujiao; Tang, Xiaoliang; Dou, Wei; Cui, Yumei; Liu, Weisheng

2018-07-19

A two-photon fluorescent probe for Cu 2+ and S 2- has been strategically prepared with naphthalimide derivative platform (NPE) covalently grafted onto the surface of magnetic core-shell Fe 3 O 4 @SiO 2 nanoparticles. The probe (NPE-Fe 3 O 4 @SiO 2 ) exhibits selective response to Cu 2+ with enhanced fluorescence and efficient separation of Cu 2+ with external magnetic field. The consequent product NPE-Fe 3 O 4 @SiO 2 -Cu of NPE-Fe 3 O 4 @SiO 2 and Cu 2+ can work as an excellent sensor for S 2- by removing Cu 2+ from the complex with fluorescence decreased, recovering the fluorescence of the probe. Therefore, the constituted Off-On-Off type fluorescence monitoring system means the probe is resumable. Moreover, the probe has been used to quantitatively detect Cu 2+ and S 2- with low detection limits, which are 0.28 μM and 0.12 μM, respectively. Furthermore, the probe shows low cytotoxicity and excellent membrane permeability, which has been successfully applied for monitoring Cu 2+ and S 2- in living cells and imaging Cu 2+ in deep-tissue with two-photon excited fluorescence. Copyright © 2018. Published by Elsevier B.V.

Engineered Aptamers to Probe Molecular Interactions on the Cell Surface

PubMed Central

Batool, Sana; Bhandari, Sanam; George, Shanell; Okeoma, Precious; Van, Nabeela; Zümrüt, Hazan E.; Mallikaratchy, Prabodhika

2017-01-01

Significant progress has been made in understanding the nature of molecular interactions on the cell membrane. To decipher such interactions, molecular scaffolds can be engineered as a tool to modulate these events as they occur on the cell membrane. To guarantee reliability, scaffolds that function as modulators of cell membrane events must be coupled to a targeting moiety with superior chemical versatility. In this regard, nucleic acid aptamers are a suitable class of targeting moieties. Aptamers are inherently chemical in nature, allowing extensive site-specific chemical modification to engineer sensing molecules. Aptamers can be easily selected using a simple laboratory-based in vitro evolution method enabling the design and development of aptamer-based functional molecular scaffolds against wide range of cell surface molecules. This article reviews the application of aptamers as monitors and modulators of molecular interactions on the mammalian cell surface with the aim of increasing our understanding of cell-surface receptor response to external stimuli. The information gained from these types of studies could eventually prove useful in engineering improved medical diagnostics and therapeutics. PMID:28850067

Oil-in-water monitoring using membrane inlet mass spectrometry.

PubMed

Brkić, Boris; France, Neil; Taylor, Stephen

2011-08-15

A membrane inlet mass spectrometry (MIMS) system has been used for detection and analysis of two types of North Sea crude oil. The system was installed on-field on the Flotta Oil Terminal (Orkney, UK). It consisted of a quadrupole mass spectrometer (QMS) connected to the capillary probe with a silicone-based membrane. The produced mass spectra and calibration plots from the MIMS instrument showed the capability to measure levels of individual hydrocarbons within crude oil in seawater. The generated mass spectra from the field tests also showed the ability to distinguish between different types of oil and to determine concentrations of toxic hydrocarbons in oil (e.g., benzene, toluene, and xylene (BTX)). The performance of the instrument at different temperatures of seawater and oil droplet sizes was also investigated. The results showed that the QMS-based MIMS system has a potential to complement existing oil-in-water (OiW) monitors by being able to detect different oil types and specific hydrocarbon concentrations with high accuracy, which are currently not supported in commercially available OiW monitors.

High-temperature fiber-optic lever microphone

NASA Technical Reports Server (NTRS)

Zuckerwar, Allan J.; Cuomo, Frank W.; Nguyen, Trung D.; Rizzi, Stephen A.; Clevenson, Sherman A.

1995-01-01

The design and construction of a fiber-optic lever microphone, capable of operating continuously at temperatures up to 538 C (1000 F) are described. The design is based on the theoretical sensitivities of each of the microphone system components, namely, a cartridge containing a stretched membrane, an optical fiber probe, and an optoelectronic amplifier. Laboratory calibrations include the pistonphone sensitivity and harmonic distortion at ambient temperature, and frequency response, background noise, and optical power transmission at both ambient and elevated temperatures. A field test in the Thermal Acoustic Fatigue Apparatus at Langley Research Center, in which the microphone was subjected to overall sound-pressure levels in the range of 130-160 dB and at temperatures from ambient to 538 C, revealed good agreement with a standard probe microphone.

Ultra-thin Oxide Membranes: Synthesis and Carrier Transport

NASA Astrophysics Data System (ADS)

Sim, Jai Sung

Self-supported freestanding membranes are films that are devoid of any underlying supporting layers. The key advantage of such structures is that, due to the lack of substrate effects - both mechanical and chemical, the true native properties of the material can be probed. This is crucial since many of the studies done on materials that are used as freestanding membranes are done as films clamped to substrates or in the bulk form. This thesis focuses on the synthesis and fabrication as well as electrical studies of free standing ultrathin < 40nm oxide membranes. It also is one of the first demonstrations for electrically probing nanoscale freestanding oxide membranes. Fabrication of such membranes is non-trivial as oxide materials are often brittle and difficult to handle. Therefore, it requires an understanding of thin plate mechanics coupled with controllable thin film deposition process. Taking things a step further, to electrically probe these membranes required design of complex device architecture and extensive optimization of nano-fabrication processes. The challenges and optimized fabrication method of such membranes are demonstrated. Three materials are probed in this study, VO2, TiO2, and CeO2. VO2 for understanding structural considerations for electronic phase change and nature of ionic liquid gating, TiO2 and CeO2 for understanding surface conduction properties and surface chemistry. The VO2 study shows shift in metal-insulator transition (MIT) temperature arising from stress relaxation and opening of the hysteresis. The ionic liquid gating studies showed reversible modulation of channel resistance and allowed distinguishing bulk process from the surface effects. Comparing the ionic liquid gating experiments to hydrogen doping experiments illustrated that ionic liquid gating can be a surface limited electrostatic effect, if the critical voltage threshold is not exceeded. TiO2 study shows creation of non-stoichiometric forms under ion milling. Utilizing focused ion beam milling, thin membranes of Ti xOy of 100-300 nm thickness have been created. TEM studies indicated polycrystallinity and presence of twins in the FIB-milled nanowalls. Compositional analysis in the transmission electron microscope also showed reduced content of oxygen, confirming non-stoichiometry. Temperature dependence of the electrical resistivity of the nanowall showed semiconducting behavior with an activation energy different from that of TiO2 single crystals and was attributed to formation of TinO2n-1 phases after FIB processing. The CeO2 study involved high temperature conductivity studies on substrate-free self-supported nano-crystalline ceria membranes up to 800 K. Increasing conductivity with oxygen partial pressure directly opposing the behavior of thin film devices 'clamped' by substrate has been observed. This illustrate that the relaxed nature of free standing membranes, and increased surface to volume ratio enables more sensitive electrical response to oxygen adsorption which could have implications for their use in oxygen storage devices, solid oxide fuel cells, and chemical sensors. The work in this thesis advances the understanding of materials in freestanding membrane form and advances fabrication techniques that have not been explored before, having implications for sensors, actuators, SOFC, memristors, and physics of quasi-2D materials.

Uniform and Janus-like nanoparticles in contact with vesicles: energy landscapes and curvature-induced forces.

PubMed

Agudo-Canalejo, Jaime; Lipowsky, Reinhard

2017-03-15

Biological membranes and lipid vesicles often display complex shapes with non-uniform membrane curvature. When adhesive nanoparticles with chemically uniform surfaces come into contact with such membranes, they exhibit four different engulfment regimes as recently shown by a systematic stability analysis. Depending on the local curvature of the membrane, the particles either remain free, become partially or completely engulfed by the membrane, or display bistability between free and completely engulfed states. Here, we go beyond stability analysis and develop an analytical theory to leading order in the ratio of particle-to-vesicle size. This theory allows us to determine the local and global energy landscapes of uniform nanoparticles that are attracted towards membranes and vesicles. While the local energy landscape depends only on the local curvature of the vesicle membrane and not on the overall membrane shape, the global energy landscape describes the variation of the equilibrium state of the particle as it probes different points along the membrane surface. In particular, we find that the binding energy of a partially engulfed particle depends on the 'unperturbed' local curvature of the membrane in the absence of the particle. This curvature dependence leads to local forces that pull the partially engulfed particles towards membrane segments with lower and higher mean curvature if the particles originate from the exterior and interior solution, respectively, corresponding to endo- and exocytosis. Thus, for partial engulfment, endocytic particles undergo biased diffusion towards the membrane segments with the lowest membrane curvature, whereas exocytic particles move towards segments with the highest curvature. The curvature-induced forces are also effective for Janus particles with one adhesive and one non-adhesive surface domain. In fact, Janus particles with a strongly adhesive surface domain are always partially engulfed which implies that they provide convenient probes for experimental studies of the curvature-induced forces that arise for complex-shaped membranes.

Functional imaging of microdomains in cell membranes.

PubMed

Duggan, James; Jamal, Ghadir; Tilley, Mark; Davis, Ben; McKenzie, Graeme; Vere, Kelly; Somekh, Michael G; O'Shea, Paul; Harris, Helen

2008-10-01

The presence of microdomains or rafts within cell membranes is a topic of intense study and debate. The role of these structures in cell physiology, however, is also not yet fully understood with many outstanding problems. This problem is partly based on the small size of raft structures that presents significant problems to their in vivo study, i.e., within live cell membranes. But the structure and dynamics as well as the factors that control the assembly and disassembly of rafts are also of major interest. In this review we outline some of the problems that the study of rafts in cell membranes present as well as describing some views of what are considered the generalised functions of membrane rafts. We point to the possibility that there may be several different 'types' of membrane raft in cell membranes and consider the factors that affect raft assembly and disassembly, particularly, as some researchers suggest that the lifetimes of rafts in cell membranes may be sub-second. We attempt to review some of the methods that offer the ability to interrogate rafts directly as well as describing factors that appear to affect their functionality. The former include both near-field and far-field optical approaches as well as scanning probe techniques. Some of the advantages and disadvantages of these techniques are outlined. Finally, we describe our own views of raft functionality and properties, particularly, concerning the membrane dipole potential, and describe briefly some of the imaging strategies we have developed for their study.

Design of quantum dot-conjugated lipids for long-term, high-speed tracking experiments on cell surfaces.

PubMed

Murcia, Michael J; Minner, Daniel E; Mustata, Gina-Mirela; Ritchie, Kenneth; Naumann, Christoph A

2008-11-12

The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking experiments on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 molar mixture of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol-2000], are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aqueous solution is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking experiments on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-molecule tracking probes in plasma membranes of several cell types. Initial tracking experiments at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-molecule imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking experiments that used larger (40 nm) Au labels, not only push the time resolution in long-time, continuous fluorescence-based single-molecule tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor experiments.

Polarization-induced local pore-wall functionalization for biosensing: from micropore to nanopore.

PubMed

Liu, Jie; Pham, Pascale; Haguet, Vincent; Sauter-Starace, Fabien; Leroy, Loïc; Roget, André; Descamps, Emeline; Bouchet, Aurélie; Buhot, Arnaud; Mailley, Pascal; Livache, Thierry

2012-04-03

The use of biological-probe-modified solid-state pores in biosensing is currently hindered by difficulties in pore-wall functionalization. The surface to be functionalized is small and difficult to target and is usually chemically similar to the bulk membrane. Herein, we demonstrate the contactless electrofunctionalization (CLEF) approach and its mechanism. This technique enables the one-step local functionalization of the single pore wall fabricated in a silica-covered silicon membrane. CLEF is induced by polarization of the pore membrane in an electric field and requires a sandwich-like composition and a conducting or semiconducting core for the pore membrane. The defects in the silica layer of the micropore wall enable the creation of an electric pathway through the silica layer, which allows electrochemical reactions to take place locally on the pore wall. The pore diameter is not a limiting factor for local wall modification using CLEF. Nanopores with a diameter of 200 nm fabricated in a silicon membrane and covered with native silica layer have been successfully functionalized with this method, and localized pore-wall modification was obtained. Furthermore, through proof-of-concept experiments using ODN-modified nanopores, we show that functionalized nanopores are suitable for translocation-based biosensing.

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

NASA Astrophysics Data System (ADS)

Heuvingh, J.; Pincet, F.; Cribier, S.

2004-07-01

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of “hemifusion” is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

Microwave-Driven Multifunctional Capability of Membrane Structures

NASA Technical Reports Server (NTRS)

Choi, Sang H.; Chu, Sang-Hyong; Song, Kyo D.; King, Glen C.

2002-01-01

A large, ultra lightweight space structure, such as solar sails and Gossamer spacecrafts, requires a distributed power source to alleviate wire networks, unlike the localized on-board power infrastructures typically found in most small spacecrafts. The concept of microwave-driven multifunctional capability for membrane structures is envisioned as the best option to alleviate the complexity associated with hard-wired control circuitry and on-board power infrastructures. A rectenna array based on a patch configuration for high voltage output was developed to drive membrane actuators, sensors, probes, or other devices. Networked patch rectenna array receives and converts microwave power into a DC power for an array of smart actuators. To use microwave power effectively, the concept of a power allocation and distribution (PAD) circuit is adopted for networking a rectenna/actuator patch array. The use of patch rectennas adds a significant amount of rigidity to membrane flexibility and they are relatively heavy. A dipole rectenna array (DRA) appears to be ideal for thin-film membrane structures, since DRA is flexible and light. Preliminary design and fabrication of PAD circuitry that consists of a few nodal elements were made for laboratory testing. The networked actuators were tested to correlate the network coupling effect, power allocation and distribution, and response time.

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

PubMed

Stock, Roberto P; Brewer, Jonathan; Wagner, Kerstin; Ramos-Cerrillo, Blanca; Duelund, Lars; Jernshøj, Kit Drescher; Olsen, Lars Folke; Bagatolli, Luis A

2012-01-01

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

A robust nanofluidic membrane with tunable zero-order release for implantable dose specific drug delivery.

PubMed

Fine, Daniel; Grattoni, Alessandro; Hosali, Sharath; Ziemys, Arturas; De Rosa, Enrica; Gill, Jaskaran; Medema, Ryan; Hudson, Lee; Kojic, Milos; Milosevic, Miljan; Brousseau Iii, Louis; Goodall, Randy; Ferrari, Mauro; Liu, Xuewu

2010-11-21

This manuscript demonstrates a mechanically robust implantable nanofluidic membrane capable of tunable long-term zero-order release of therapeutic agents in ranges relevant for clinical applications. The membrane, with nanochannels as small as 5 nm, allows for the independent control of both dosage and mechanical strength through the integration of high-density short nanochannels parallel to the membrane surface with perpendicular micro- and macrochannels for interfacing with the ambient solutions. These nanofluidic membranes are created using precision silicon fabrication techniques on silicon-on-insulator substrates enabling exquisite control over the monodispersed nanochannel dimensions and surface roughness. Zero-order release of analytes is achieved by exploiting molecule to surface interactions which dominate diffusive transport when fluids are confined to the nanoscale. In this study we investigate the nanofluidic membrane performance using custom diffusion and gas testing apparatuses to quantify molecular release rate and process uniformity as well as mechanical strength using a gas based burst test. The kinetics of the constrained zero-order release is probed with molecules presenting a range of sizes, charge states, and structural conformations. Finally, an optimal ratio of the molecular hydrodynamic diameter to the nanochannel dimension is determined to assure zero-order release for each tested molecule.

Aptamer based surface enhanced Raman scattering detection of vasopressin using multilayer nanotube arrays

PubMed Central

Huh, Yun Suk; Erickson, David

2009-01-01

Here we present an optofluidic surface enhanced Raman spectroscopy (SERS) device for on-chip detection of vasopressin using an aptamer based binding assay. To create the SERS-active substrate, densely packed, 200 nm diameter, metal nanotube arrays were fabricated using an anodized alumina nanoporous membrane as a template for shadow evaporation. We explore the use of both single layer Au structures and multilayer Au/Ag/Au structures and also demonstrate a facile technique for integrating the membranes with all polydimethylsiloxane (PDMS) microfluidic devices. Using the integrated device, we demonstrate a linear response in the main detection peak intensity to solution phase concentration and a limit of detection on the order of 5.2 μU/mL. This low limit of detection is obtained with device containing the multilayer SERS substrate which we show exhibits a stronger Raman enhancement while maintaining biocompatibility and ease or surface reactivity with the capture probe. PMID:19857952

A novel microfluidics-based method for probing weak protein-protein interactions.

PubMed

Tan, Darren Cherng-wen; Wijaya, I Putu Mahendra; Andreasson-Ochsner, Mirjam; Vasina, Elena Nikolaevna; Nallani, Madhavan; Hunziker, Walter; Sinner, Eva-Kathrin

2012-08-07

We report the use of a novel microfluidics-based method to detect weak protein-protein interactions between membrane proteins. The tight junction protein, claudin-2, synthesised in vitro using a cell-free expression system in the presence of polymer vesicles as membrane scaffolds, was used as a model membrane protein. Individual claudin-2 molecules interact weakly, although the cumulative effect of these interactions is significant. This effect results in a transient decrease of average vesicle dispersivity and reduction in transport speed of claudin-2-functionalised vesicles. Polymer vesicles functionalised with claudin-2 were perfused through a microfluidic channel and the time taken to traverse a defined distance within the channel was measured. Functionalised vesicles took 1.19 to 1.69 times longer to traverse this distance than unfunctionalised ones. Coating the channel walls with protein A and incubating the vesicles with anti-claudin-2 antibodies prior to perfusion resulted in the functionalised vesicles taking 1.75 to 2.5 times longer to traverse this distance compared to the controls. The data show that our system is able to detect weak as well as strong protein-protein interactions. This system offers researchers a portable, easily operated and customizable platform for the study of weak protein-protein interactions, particularly between membrane proteins.

[The effect of alpha-tocopherol and ionol on the physical structure of the membranes of rat liver microsomes under conditions of antioxidant insufficiency].

PubMed

Gubskiĭ, Iu I; Boldeskul, A E; Primak, R G; Zadorina, O V

1989-01-01

Physiochemical conformity of the alpha-tocopherol interaction with hepatic microsomal membranes has been studied by means of fluorescent probes (pyrene and 1-anilinonaphthalene-8-sulphonate). The microsomal membrane microviscosity is shown to sharply decrease under conditions of the antioxidant deficiency with vitamin E expelled into animals normalizes microviscosity, but feebly influences the microsomal surface charge. Microcalorimetry has been used to establish that penetration of tocopherol into microsomal membranes was accompanied by the exothermic effect.

Evaluation of the efficacy of 100% Type-I collagen membrane of bovine origin in the treatment of human gingival recession: A clinical study

PubMed Central

Soni, Nitin; Sikri, Poonam; Kapoor, Daljit; Soni, Bhavita Wadhwa; Jain, Rachna

2014-01-01

Background: Various treatment modalities have been devised for gingival recession, which is one of the most common signs of periodontal disease. The present study evaluates the efficacy of bioresorbable 100% type I collagen membrane of bovine origin in the treatment of human gingival recession. Materials and Methods: Twenty cases of Miller's class I or class II localized gingival recession defects on the facial surface were treated with 100% type I collagen membrane of bovine origin in conjunction with coronally positioned flap. Pre-operative and post-operative assessments were performed with respect to probing pocket depth, clinical attachment level and clinical recession at 12, 24 and 36 weeks. The data thus collected were analyzed statistically. Results: Statistically significant improvement based on Student's t test was found in all the three clinical parameters. Conclusion: Bioresorbable 100% type I collagen membrane of bovine origin has given inspiring results in the treatment of human gingival recession defects, thereby justifying the use of this material wherever indicated. PMID:25565742

Functional Na+ Channels in Cell Adhesion probed by Transistor Recording

PubMed Central

Schmidtner, Markus; Fromherz, Peter

2006-01-01

Cell membranes in a tissue are in close contact to each other, embedded in the extracellular matrix. Standard electrophysiological methods are not able to characterize ion channels under these conditions. Here we consider the area of cell adhesion on a solid substrate as a model system. We used HEK 293 cells cultured on fibronectin and studied the activation of NaV1.4 sodium channels in the adherent membrane with field-effect transistors in a silicon substrate. Under voltage clamp, we compared the transistor response with the whole-cell current. We observed that the extracellular voltage in the cell-chip contact was proportional to the total membrane current. The relation was calibrated by alternating-current stimulation. We found that Na+ channels are present in the area of cell adhesion on fibronectin with a functionality and a density that is indistinguishable from the free membrane. The experiment provides a basis for studying selective accumulation and depletion of ion channels in cell adhesion and also for a development of cell-based biosensoric devices and neuroelectronic systems. PMID:16227504

All-Atom Molecular Dynamics-Based Analysis of Membrane-Stabilizing Copolymer Interactions with Lipid Bilayers Probed under Constant Surface Tensions.

PubMed

Houang, Evelyne M; Bates, Frank S; Sham, Yuk Y; Metzger, Joseph M

2017-11-30

An all-atom phospholipid bilayer and triblock copolymer model was developed for molecular dynamics (MD) studies. These were performed to investigate the mechanism of interaction between membrane-stabilizing triblock copolymer P188 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers under applied lateral surface tension (γ) to model membrane mechanical stress. Results showed that P188 insertion is driven by the hydrophobic poly(propylene oxide) (PPO) core and dependent on bilayer area per lipid. Moreover, insertion of P188 increased the bilayer's resistance to mechanical rupture, as observed by a significant increase in the absolute lateral pressure required to disrupt the bilayer. To further investigate the specific chemical features of P188 underlying membrane stabilizer function, a series of MD simulations with triblock copolymers of the same class as P188 but of varying chemical composition and sizes were performed. Results showed that triblock copolymer insertion into the lipid bilayer is dependent on overall copolymer hydrophobicity, with higher copolymer hydrophobicity requiring a reduced bilayer area per lipid ratio for insertion. Further analysis revealed that the effect of copolymer insertion on membrane mechanical integrity was also dependent on hydrophobicity. Here, P188 insertion significantly increased the absolute apparent lateral pressure required to rupture the POPC bilayer, thereby protecting the membrane against mechanical stress. In marked contrast, highly hydrophobic copolymers decreased the lateral pressure necessary for membrane rupture and thus rendering the membrane significantly more susceptible to mechanical stress. These new in silico findings align with recent experimental findings using synthetic lipid bilayers and in muscle cells in vitro and mouse models in vivo. Collectively, these data underscore the importance of PEO-PPO-PEO copolymer chemical composition in copolymer-based muscle membrane stabilization in vitro and in vivo. All-atom modeling with MD simulations holds promise for investigating novel copolymers with enhanced membrane interacting properties.

Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-Color Live Cell Imaging and Cell Damage Diagnosis.

PubMed

Chao, Xi-Juan; Wang, Kang-Nan; Sun, Li-Li; Cao, Qian; Ke, Zhuo-Feng; Cao, Du-Xia; Mao, Zong-Wan

2018-04-25

Studies on the development of fluorescent organic molecules with different emission colors for imaging of organelles and their biomedical application are gaining lots of focus recently. Here, we report two cationic organochalcogens 1 and 2, both of which exhibit very weak green emission (Φ 1 = 0.12%; Φ 2 = 0.09%) in dilute solution as monomers, but remarkably enhanced green emission upon interaction with nucleic acids and large red-shifted emission in aggregate state by the formation of excimers at high concentration. More interestingly, the monomer emission and excimer-like emission can be used for dual color imaging of different organelles. Upon passively diffusing into cells, both probes selectively stain nucleoli with strong green emission upon 488 nm excitation, whereas upon 405 nm excitation, a completely different stain pattern by staining lysosomes (for 1) or mitochondria (for 2) with distinct red emission is observed because of the highly concentrated accumulation in these organelles. Studies on the mechanism of the accumulation in lysosomes (for 1) or mitochondria (for 2) found that the accumulations of the probes are dependent on the membrane permeabilization, which make the probes have great potential in diagnosing cell damage by sensing lysosomal or mitochondrial membrane permeabilization. The study is demonstrative, for the first time, of two cationic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell based on monomer and excimer-like emission, respectively, and more importantly, for diagnosing cell damage.

Specific PET Imaging Probes for Early Detection of Prostate Cancer Metastases

DTIC Science & Technology

2010-05-01

penetrating cell membranes. In one of our studies using such a peptide to deliver a therapeutic moiety to various prostate cancer cell lines, we...to exploit this group of peptides for the early detection of prostate tumor metastases. Promisingly, in our preliminary studies , th e peptide lab...cancer. Based on one of our studies using a polyarginine (NH2GR11) to deliver a therapeutic moiety to various prostate cancer cell lines, we hypothesize

Hetero-bivalent Imaging Agents for Simultaneous Targeting Prostate-Specific Membrane Antigen (PSMA) and Hepsin

DTIC Science & Technology

2012-09-01

functions of PSMA for PCa, we have designed and prepared PSMA-based imaging probes to evaluate its biological activity in vivo in a variety of...nucleophilic functional group to be coupled with radionuclides, i.e., 125I or 18F, or optical dyes. C. Accomplishments in Year 2 1...position of the lysine or N-terminus isoleucine with the imaging prosthetic groups such as optical dyes and radionuclides. The alloc group was

Optical Fiber Sensing Using Quantum Dots

PubMed Central

Jorge, Pedro; Martins, Manuel António; Trindade, Tito; Santos, José Luís; Farahi, Faramarz

2007-01-01

Recent advances in the application of semiconductor nanocrystals, or quantum dots, as biochemical sensors are reviewed. Quantum dots have unique optical properties that make them promising alternatives to traditional dyes in many luminescence based bioanalytical techniques. An overview of the more relevant progresses in the application of quantum dots as biochemical probes is addressed. Special focus will be given to configurations where the sensing dots are incorporated in solid membranes and immobilized in optical fibers or planar waveguide platforms. PMID:28903308

Novel pH-Sensitive Lipid Based Exo-Endocytosis Tracers Reveal Fast Intermixing of Synaptic Vesicle Pools

PubMed Central

Kahms, Martin; Klingauf, Jürgen

2018-01-01

Styryl dyes and genetically encoded pH-sensitive fluorescent proteins like pHluorin are well-established tools for the optical analysis of synaptic vesicle (SV) recycling at presynaptic boutons. Here, we describe the development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes. These new optical tracers allow a pure read out of membrane turnover during synaptic activity and visualization of multiple rounds of stimulation-dependent SV recycling without genetic perturbation. Measuring the incorporation efficacy of different dye-labeled lipids into budding SVs, we did not observe an enrichment of lipids with affinity for liquid ordered membrane domains. But most importantly, we found no evidence for a static segregation of SVs into recycling and resting pools. A small but significant fraction of SVs that is reluctant to release during a first round of evoked activity can be exocytosed during a second bout of stimulation, showing fast intermixing of SV pools within seconds. Furthermore, we found that SVs recycling spontaneously have a higher chance to re-occupy release sites than SVs recycling during high-frequency evoked activity. In summary, our data provide strong evidence for a highly dynamic and use-dependent control of the fractions of releasable or resting SVs. PMID:29456492

Relative quantification of membrane-associated calcium in red spruce mesophyll cells

Treesearch

Catherine H. Borer; Paul Schaberg; Jonathan R. Cumming

1997-01-01

We describe a method for localizing and comparing relative amounts of plasma membrane-associated calcium ions (mCa) in complex tissues and verify the procedure for mesophyll cells of red spruce (Picea rubens Sarg.) needles. This technique incorporates epifluorescence microscopy using the fluorescent probe chlorotetracycline (CTC) with computer image...

Estimation of the Asymmetrical Arrangement of Plasma Membrane Aminophospholipids. An Experimental Assay for Students of Biochemistry and Molecular Biology.

ERIC Educational Resources Information Center

Sanchez-Yague, J.; And Others

1987-01-01

Describes an experiment to discover the topology of plasma membrane aminophospholipids (phosphatidylethanolamine and phosphatidylserine) using whole platelets and trinitrobenzene sulfonate (TNBS) as a probe. Reports changes in phospholipid distribution during platelet activation with simultaneous action of thrombia and collagen. Details the…

Nanoscale diffractive probing of strain dynamics in ultrafast transmission electron microscopy

PubMed Central

Feist, Armin; Rubiano da Silva, Nara; Liang, Wenxi; Ropers, Claus; Schäfer, Sascha

2018-01-01

The control of optically driven high-frequency strain waves in nanostructured systems is an essential ingredient for the further development of nanophononics. However, broadly applicable experimental means to quantitatively map such structural distortion on their intrinsic ultrafast time and nanometer length scales are still lacking. Here, we introduce ultrafast convergent beam electron diffraction with a nanoscale probe beam for the quantitative retrieval of the time-dependent local deformation gradient tensor. We demonstrate its capabilities by investigating the ultrafast acoustic deformations close to the edge of a single-crystalline graphite membrane. Tracking the structural distortion with a 28-nm/700-fs spatio-temporal resolution, we observe an acoustic membrane breathing mode with spatially modulated amplitude, governed by the optical near field structure at the membrane edge. Furthermore, an in-plane polarized acoustic shock wave is launched at the membrane edge, which triggers secondary acoustic shear waves with a pronounced spatio-temporal dependency. The experimental findings are compared to numerical acoustic wave simulations in the continuous medium limit, highlighting the importance of microscopic dissipation mechanisms and ballistic transport channels. PMID:29464187

Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks

NASA Astrophysics Data System (ADS)

Bayburt, Timothy H.; Sligar, Stephen G.

2002-05-01

The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

Evaluation of toxic agent effects on lung cells by fiber evanescent wave spectroscopy.

PubMed

Lucas, Pierre; Le Coq, David; Juncker, Christophe; Collier, Jayne; Boesewetter, Dianne E; Boussard-Plédel, Catherine; Bureau, Bruno; Riley, Mark R

2005-01-01

Biochemical changes in living cells are detected using a fiber probe system composed of a single chalcogenide fiber acting as both the sensor and transmission line for infrared optical signals. The signal is collected via evanescent wave absorption along the tapered sensing zone of the fiber. We spectroscopically monitored the effects of the surfactant Triton X-100, which serves as a toxic agent simulant on a transformed human lung carcinoma type II epithelial cell line (A549). We observe spectral changes between 2800-3000 cm(-1) in four absorptions bands, which are assigned to hydrocarbon vibrations of methylene and methyl groups in membrane lipids. Comparison of fiber and transmission spectra shows that the present technique allows one to locally probe the cell plasma membrane in the lipid spectral region. These optical responses are correlated with cellular metabolic activity measurements and LDH (lactate dehydrogenase) release assays that indicate a loss of cellular function and membrane integrity as would be expected in response to the membrane solubilizing Triton. The spectroscopic technique shows a significantly greater detection resolution in time and concentration.

Nanoscale diffractive probing of strain dynamics in ultrafast transmission electron microscopy.

PubMed

Feist, Armin; Rubiano da Silva, Nara; Liang, Wenxi; Ropers, Claus; Schäfer, Sascha

2018-01-01

The control of optically driven high-frequency strain waves in nanostructured systems is an essential ingredient for the further development of nanophononics. However, broadly applicable experimental means to quantitatively map such structural distortion on their intrinsic ultrafast time and nanometer length scales are still lacking. Here, we introduce ultrafast convergent beam electron diffraction with a nanoscale probe beam for the quantitative retrieval of the time-dependent local deformation gradient tensor. We demonstrate its capabilities by investigating the ultrafast acoustic deformations close to the edge of a single-crystalline graphite membrane. Tracking the structural distortion with a 28-nm/700-fs spatio-temporal resolution, we observe an acoustic membrane breathing mode with spatially modulated amplitude, governed by the optical near field structure at the membrane edge. Furthermore, an in-plane polarized acoustic shock wave is launched at the membrane edge, which triggers secondary acoustic shear waves with a pronounced spatio-temporal dependency. The experimental findings are compared to numerical acoustic wave simulations in the continuous medium limit, highlighting the importance of microscopic dissipation mechanisms and ballistic transport channels.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

PubMed

Sakurai, Chiye; Itakura, Makoto; Kinoshita, Daiki; Arai, Seisuke; Hashimoto, Hitoshi; Wada, Ikuo; Hatsuzawa, Kiyotaka

2018-05-17

SNAP-23 is a plasma membrane-localized SNARE protein involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation specific-antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the non-phosphorylatable S95A or the phospho-mimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D-overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N-termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Co-expression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ-activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine Fluorophore into a Phospholipid Bilayer: Complementary Use of Fluorescence Quenching Studies and Molecular Dynamics Simulations

PubMed Central

Kyrychenko, Alexander; Sevriukov, Igor Yu.; Syzova, Zoya A.; Ladokhin, Alexey S.; Doroshenko, Andrey O.

2014-01-01

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from -6.5 to -7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP. PMID:21211898

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.

Na+/H+ and K+/H+ Antiport in Root Membrane Vesicles Isolated from the Halophyte Atriplex and the Glycophyte Cotton 1

PubMed Central

Hassidim, Miriam; Braun, Yael; Lerner, Henri R.; Reinhold, Leonora

1990-01-01

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [14C]methylamine. Evidence is presented for the operation of Na+/H+ and K+/H+ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H+-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl−, using the ΔΨ probe SCN−. Cation/H+ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H+-ATPase; coupling of the opposed cation and H+ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na+/H+ and K+/H+ antiport in Atriplex. Moreover, additive effects are obtained when Na+ is supplied together with saturating concentrations of K+, and vice versa, suggesting that separate antiporters for Na+ and for K+ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast. PMID:16667918

Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton.

PubMed

Hassidim, M; Braun, Y; Lerner, H R; Reinhold, L

1990-12-01

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the DeltapH probe [(14)C]methylamine. Evidence is presented for the operation of Na(+)/H(+) and K(+)/H(+) antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H(+)-ATPase or by imposing an artificial pH gradient) brings about dissipation of the DeltapH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl(-), using the DeltaPsi probe SCN(-). Cation/H(+) exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H(+)-ATPase; coupling of the opposed cation and H(+) fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na(+)/H(+) and K(+)/H(+) antiport in Atriplex. Moreover, additive effects are obtained when Na(+) is supplied together with saturating concentrations of K(+), and vice versa, suggesting that separate antiporters for Na(+) and for K(+) may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.

Principles and biophysical applications of single particle super-localization and rotational tracking

NASA Astrophysics Data System (ADS)

Gu, Yan

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.

Principles and biophysical applications of single particle super-localization and rotational tracking

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

Gu, Yan

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. Wemore » found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.« less

Pore spanning lipid bilayers on silanised nanoporous alumina membranes

NASA Astrophysics Data System (ADS)

Md Jani, Abdul M.; Zhou, Jinwen; Nussio, Matthew R.; Losic, Dusan; Shapter, Joe G.; Voelcker, Nicolas H.

2008-12-01

The preparation of bilayer lipid membranes (BLMs) on solid surfaces is important for many studies probing various important biological phenomena including the cell barrier properties, ion-channels, biosensing, drug discovery and protein/ligand interactions. In this work we present new membrane platforms based on suspended BLMs on nanoporous anodic aluminium oxide (AAO) membranes. AAO membranes were prepared by electrochemical anodisation of aluminium foil in 0.3 M oxalic acid using a custom-built etching cell and applying voltage of 40 V, at 1oC. AAO membranes with controlled diameter of pores from 30 - 40 nm (top of membrane) and 60 -70 nm (bottom of membrane) were fabricated. Pore dimensions have been confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). AAO membranes were chemically functionalised with 3-aminopropyltriethoxysilane (APTES). Confirmation of the APTES attachment to the AAO membrane was achieved by means of infrared spectroscopy, X-ray photoelectron spectroscopy and contact angle measurements. The Fourier transform infrared (FTIR) spectra of functionalised membranes show several peaks from 2800 to 3000 cm-1 which were assigned to symmetric and antisymmetric CH2 bands. XPS data of the membrane showed a distinct increase in C1s (285 eV), N1s (402 eV) and Si2p (102 eV) peaks after silanisation. The water contact angle of the functionalised membrane was 80o as compared to 20o for the untreated membrane. The formation of BLMs comprising dioleoyl-phosphatidylserine (DOPS) on APTESmodified AAO membranes was carried using the vesicle spreading technique. AFM imaging and force spectroscopy was used to characterise the structural and nanomechanical properties of the suspended membrane. This technique also confirmed the stability of bilayers on the nanoporous alumina support for several days. Fabricated suspended BLMs on nanoporous AAO hold promise for the construction of biomimetic membrane architectures with embedded transmembrane proteins.

Development and application of a ruthenium(II) complex-based photoluminescent and electrochemiluminescent dual-signaling probe for nitric oxide.

PubMed

Zhang, Wenzhu; Zhang, Jingmei; Zhang, Hailei; Cao, Liyan; Zhang, Run; Ye, Zhiqiang; Yuan, Jingli

2013-11-15

A ruthenium(II) complex, [Ru(bpy)2(DA-phen)](PF6)2 (bpy: 2,2'-bipyridine; DA-phen: 5,6-diamino-1,10-phenanthroline), has been developed as a photoluminescent (PL) and electrochemiluminescent (ECL) dual-signaling probe for the highly sensitive and selective detection of nitric oxide (NO) in aqueous and biological samples. Due to the presence of electron transfer process from diamino group to the excited-state of the Ru(II) complex, the PL and ECL intensities of the probe are very weak. After the probe was reacted with NO in physiological pH aqueous media under aerobic conditions to afford its triazole derivative, [Ru(bpy)2(TA-phen)](2+) (TA-phen: 5,6-triazole-1,10-phenanthroline), the electron transfer process was inhibited, so that the PL and ECL efficiency of the Ru(II) complex was remarkably increased. The PL and ECL responses of the probe to NO in physiological pH media are highly sensitive with the detection limits at low micromolar concentration level, and highly specific without the interferences of other reactive oxygen/nitrogen species (ROS/RNS) and metal ions. Moreover, the probe has good cell-membrane permeability, and can be rapidly transferred into living cells for trapping the intracellular NO molecules. These features enabled the probe to be successfully used for the monitoring of the endogenous NO production in living biological cell and tissue samples with PL and ECL dual-modes. Copyright © 2013 Elsevier B.V. All rights reserved.

Exploring the Spatiotemporal Organization of Membrane Proteins in Living Plant Cells.

PubMed

Wang, Li; Xue, Yiqun; Xing, Jingjing; Song, Kai; Lin, Jinxing

2018-04-29

Plasma membrane proteins have important roles in transport and signal transduction. Deciphering the spatiotemporal organization of these proteins provides crucial information for elucidating the links between the behaviors of different molecules. However, monitoring membrane proteins without disrupting their membrane environment remains difficult. Over the past decade, many studies have developed single-molecule techniques, opening avenues for probing the stoichiometry and interactions of membrane proteins in their native environment by providing nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we assess recent progress in the development of labeling and imaging technology for membrane protein analysis. We focus in particular on several single-molecule techniques for quantifying the dynamics and assembly of membrane proteins. Finally, we provide examples of how these new techniques are advancing our understanding of the complex biological functions of membrane proteins.

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.

Determination of excitation profile and dielectric function spatial nonuniformity in porous silicon by using WKB approach.

PubMed

He, Wei; Yurkevich, Igor V; Canham, Leigh T; Loni, Armando; Kaplan, Andrey

2014-11-03

We develop an analytical model based on the WKB approach to evaluate the experimental results of the femtosecond pump-probe measurements of the transmittance and reflectance obtained on thin membranes of porous silicon. The model allows us to retrieve a pump-induced nonuniform complex dielectric function change along the membrane depth. We show that the model fitting to the experimental data requires a minimal number of fitting parameters while still complying with the restriction imposed by the Kramers-Kronig relation. The developed model has a broad range of applications for experimental data analysis and practical implementation in the design of devices involving a spatially nonuniform dielectric function, such as in biosensing, wave-guiding, solar energy harvesting, photonics and electro-optical devices.

Structural correlation between lipophilicity and lipopolysaccharide-sequestering activity in spermine-sulfonamide analogs.

PubMed

Burns, Mark R; Jenkins, Scott A; Vermeulen, Nicolas M; Balakrishna, Rajalakshmi; Nguyen, Thuan B; Kimbrell, Matthew R; David, Sunil A

2006-12-15

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria, and play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. We had previously defined the pharmacophore necessary for small molecules to specifically bind and neutralize this complex carbohydrate. A series of aryl and aliphatic spermine-sulfonamide analogs were synthesized and tested in a series of binding and cell-based assays in order to probe the effect of lipophilicity on sequestration ability. A strong correlation was indeed found, supporting the hypothesis that endotoxin-neutralizing ability involves a lipophilic or membrane attachment event. The research discussed herein may be useful for the design of additional carbohydrate recognizing molecules and endotoxin-neutralizing drugs.

Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules

PubMed Central

Rayan, Gamal; Guet, Jean-Erik; Taulier, Nicolas; Pincet, Frederic; Urbach, Wladimir

2010-01-01

This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies. Initially, we discuss the lateral diffusion of membrane proteins, as measured by FRAP. Then, we talk about the use of FRAP to probe interactions between membrane proteins by obtaining fundamental information such as geometry and stoichiometry of the interacting complex. Afterwards, we discuss some applications of FRAP at the cellular level as well as the level of organisms. We conclude by comparing diffusion coefficients obtained by FRAP and several other alternative methods. PMID:22219695

Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers.

PubMed

Sarmento, M J; Prieto, M; Fernandes, Fábio

2012-11-01

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of phototoxicity of dendritic porphyrin-based phosphorescent oxygen probes: an in vitro study†

PubMed Central

Lebedev, Artem Y.; Marchi, Enrico; Yuan, Min; Esipova, Tatiana V.; Bergamini, Giacomo; Wilson, David F.

2013-01-01

Biological oxygen measurements by phosphorescence quenching make use of exogenous phosphorescent probes, which are introduced directly into the medium of interest (e.g. blood or interstitial fluid) where they serve as molecular sensors for oxygen. The byproduct of the quenching reaction is singlet oxygen, a highly reactive species capable of damaging biological tissue. Consequently, potential probe phototoxicity is a concern for biological applications. Herein, we compared the ability of polyethyleneglycol (PEG)-coated Pd tetrabenzoporphyrin (PdTBP)-based dendritic nanoprobes of three successive generations to sensitize singlet oxygen. It was found that the size of the dendrimer has practically no effect on the singlet oxygen sensitization efficiency in spite of the strong attenuation of the triplet quenching rate with an increase in the dendrimer generation. This unexpected result is due to the fact that the lifetime of the PdTBP triplet state in the absence of oxygen increases with dendritic generation, thus compensating for the concomitant decrease in the rate of quenching. Nevertheless, in spite of their ability to sensitize singlet oxygen, the phosphorescent probes were found to be non-phototoxic when compared with the commonly used photodynamic drug Photofrin in a standard cell-survival assay. The lack of phototoxicity is presumably due to the inability of PEGylated probes to associate with cell surfaces and/or penetrate cellular membranes. In contrast, conventional photosensitizers bind to cell components and act by generating singlet oxygen inside or in the immediate vicinity of cellular organelles. Therefore, PEGylated dendritic probes are safe to use for tissue oxygen measurements as long as the light doses are less than or equal to those commonly employed in photodynamic therapy. PMID:21409208

Visualization of hormone binding proteins in vivo based on Mn-doped CdTe QDs

NASA Astrophysics Data System (ADS)

Liu, Fang fei; Yu, Ying; Lin, Bi xia; Hu, Xiao gang; Cao, Yu juan; Wu, Jian zhong

2014-10-01

Daminozide (B9) is a growth inhibitor with important regulatory roles in plant growth and development. Locating and quantifying B9-binding proteins in plant tissues will assist in investigating the mechanism behind the signal transduction of B9. In this study, red fluorescent Mn-doped CdTe quantum dots (CdTeMn QDs) were synthesized by a high-temperature hydrothermal process. Since CdTeMn QDs possess a maximum fluorescence emission peak at 610 nm, their fluorescence properties are more stable than those of CdTe QDs. A B9-CdTeMn probe was synthesized by coupling B9 with CdTeMn QDs. The fluorescence intensity of the probe is double that of CdTeMn QDs; its fluorescence stability is also superior under different ambient conditions. The probe retains the biological activity of B9 and is unaffected by interference from the green fluorescent protein present in plants. Therefore, we used this probe to label B9-binding proteins selectively in root tissue sections of mung bean seedlings. These proteins were observed predominantly on the surfaces of the cell membranes of the cortex and epidermal parenchyma.

Probing phospholipase a(2) with fluorescent phospholipid substrates.

PubMed

Wichmann, Oliver; Gelb, Michael H; Schultz, Carsten

2007-09-03

The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.

Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation

NASA Technical Reports Server (NTRS)

Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

1997-01-01

Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.

Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation.

PubMed

Winter, H; Huber, J L; Huber, S C

1997-12-29

Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.

Low-frequency ESR studies on permeable and impermeable deuterated nitroxyl radicals in corn oil solution.

PubMed

David Jebaraj, D; Utsumi, Hideo; Milton Franklin Benial, A

2018-04-01

Low-frequency electron spin resonance studies were performed for 2 mM concentration of deuterated permeable and impermeable nitroxyl spin probes, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl and 3-carboxy-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy in pure water and various concentrations of corn oil solution. The electron spin resonance parameters such as the line width, hyperfine coupling constant, g factor, rotational correlation time, permeability, and partition parameter were estimated. The broadening of line width was observed for nitroxyl radicals in corn oil mixture. The rotational correlation time increases with increasing concentration of corn oil, which indicates the less mobile nature of spin probe in corn oil mixture. The membrane permeability and partition parameter values were estimated as a function of corn oil concentration, which reveals that the nitroxyl radicals permeate equally into the aqueous phase and oil phase at the corn oil concentration of 50%. The electron spin resonance spectra demonstrate the permeable and impermeable nature of nitroxyl spin probes. From these results, the corn oil concentration was optimized as 50% for phantom studies. In this work, the corn oil and pure water mixture phantom models with various viscosities correspond to plasma membrane, and whole blood membrane with different hematocrit levels was studied for monitoring the biological characteristics and their interactions with permeable nitroxyl spin probe. These results will be useful for the development of electron spin resonance and Overhauser-enhanced magnetic resonance imaging modalities in biomedical applications. Copyright © 2017 John Wiley & Sons, Ltd.

Response of biomembrane domains to external stimuli

NASA Astrophysics Data System (ADS)

Urbancic, Iztok

To enrich our knowledge about membrane domains, new measurement techniques with extended spatial and temporal windows are being vigorously developed by combining various approaches. Following such efforts of the scientific community, we set up fluorescence microspectroscopy (FMS), bridging two well established methods: fluorescence microscopy, which enables imaging of the samples with spatial resolution down to 200 nm, and fluorescence spectroscopy that provides molecular information of the environment at nanometer and nanosecond scale. The combined method therefore allows us to localize this type of information with the precision suitable for studying various cellular structures. Faced with weak available fluorescence signals, we have put considerable efforts into optimization of measurement processes and analysis of the data. By introducing a novel acquisition scheme and by fitting the data with a mathematical model, we preserved the spectral resolution, characteristic for spectroscopic measurements of bulk samples, also at microscopic level. We have at the same time overcome the effects of photobleaching, which had previously considerably distorted the measured spectral lineshape of photosensitive dyes and consequently hindered the reliability of FMS. Our new approach has therefore greatly extended the range of applicable environmentally sensitive probes, which can now be designed to better accommodate the needs of each particular experiment. Moreover, photobleaching of fluorescence signal can now even be exploited to obtain new valuable information about molecular environment of the probes, as bleaching rates of certain probes also depend on physical and chemical properties of the local surroundings. In this manner we increased the number of available spatially localized spectral parameters, which becomes invaluable when investigating complex biological systems that can only be adequately characterized by several independent variables. Applying the developed method FMS to several model membrane systems as well as to living cells, we showed that we can reliably detect the differences in lipid phases and membrane domains upon changes of temperature or biochemical composition. A 1--3 nm spectral shift of probes NBD and Laurdan due to different local polarity was sufficient to clearly distinguish individual vesicles in gel, liquid ordered, or liquid disordered lipid phase that had been prepared from different lipid mixtures. The results were corroborated by observations of phase transition of individual liposomes from gel to liquid disordered phase upon controlled heating of the sample by a heating slide or by a focused infrared laser beam of optical tweezers. The spectral and spatial resolution of FMS were preserved also when observing more complex biological samples, such as mixtures of liposomes and cells, showing that our results were not affected by signals of autofluorescence and growth medium, which often obstruct other fluorescence measurements. The robustness of the method allowed us to identify the delivery mechanism of a cancerostatic drug into human breast cancer cells by lipid nanoparticles. Small spectral shifts of an environment-sensitive dye revealed that the membranes of drug-carrying liposomes fuse with the membranes of cancer cells, delivering the therapeutic substance into the target. Our findings pave the way towards new biomedical approaches for more efficient treatment of the gravest maladies of our time. Furthermore, we upgraded FMS by analyzing the polarization of emitted fluorescence, which is related to the orientational order of dyes' dipoles in the membrane and therefore also to molecular conformations of the probes. The combination of spectral and polarized detection enabled us to provide the first direct experimental evidence that some of the most widely used environment-sensitive membrane probes -- NBD-labelled phospholipids -- undertake various conformations that coexist at distances below optical spatial resolution. Developing a mathematical model, we additionally characterized these conformations and determined their relative portions. We found that they are greatly affected by high concentration of cholesterol, which forces the probes into a different conformation due to its rigid planar chemical structure that favors tight packing of neighboring molecules. (Abstract shortened by UMI.)

Probing nanomechanical interaction at the interface between biological membrane and potentially toxic chemical.

PubMed

Lim, Chanoong; Park, Sohee; Park, Jinwoo; Ko, Jina; Lee, Dong Woog; Hwang, Dong Soo

2018-04-12

Various xenobiotics interact with biological membranes, and precise evaluations of the molecular interactions between them are essential to foresee the toxicity and bioavailability of existing or newly synthesized molecules. In this study, surface forces apparatus (SFA) measurement and Langmuir trough based tensiometry are performed to reveal nanomechanical interaction mechanisms between potential toxicants and biological membranes for ex vivo toxicity evaluation. As a toxicant, polyhexamethylene guanidine (PHMG) was selected because PHMG containing humidifier disinfectant and Vodka caused lots of victims in both S. Korea and Russia, respectively, due to the lack of holistic toxicity evaluation of PHMG. Here, we measured strong attraction (Wad ∼4.2 mJ/m 2 ) between PHMG and head group of biological membranes while no detectable adhesion force between the head group and control molecules was measured. Moreover, significant changes in π-A isotherm of 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) monolayers were measured upon PHMG adsorption. These results indicate PHMG strongly binds to hydrophilic group of lipid membranes and alters the structural and phase behavior of them. More importantly, complementary utilization of SFA and Langmuir trough techniques are found to be useful to predict the potential toxicity of a chemical by evaluating the molecular interaction with biological membranes, the primary protective barrier for living organisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Investigating Hydrophilic Pores in Model Lipid Bilayers using Molecular Simulations: Correlating Bilayer Properties with Pore Formation Thermodynamics

PubMed Central

Hu, Yuan; Sinha, Sudipta Kumar

2015-01-01

Cell-penetrating and antimicrobial peptides show remarkable ability to translocate across physiological membranes. Along with factors such as electric potential induced-perturbations of membrane structure and surface tension effects, experiments invoke pore-like membrane configurations during the solute transfer process into vesicles and cells. The initiation and formation of pores are associated with a non-trivial free energy cost, thus necessitating consideration of the factors associated with pore formation and attendant free energetics. Due to experimental and modeling challenges related to the long timescales of the translocation process, we use umbrella-sampling molecular dynamics simulations with a lipid-density based order parameter to investigate membrane pore-formation free energy employing Martini coarse-grained models. We investigate structure and thermodynamic features of the pore in 18 lipids spanning a range of head-groups, charge states, acyl chain lengths and saturation. We probe the dependence of pore-formation barriers on area per lipid, lipid bilayer thickness, membrane bending rigidities in three different lipid classes. The pore formation free energy in pure bilayers and peptide translocating scenarios are significantly coupled with bilayer thickness. Thicker bilayers require more reversible work to create pores. Pore formation free energy is higher in peptide-lipid systems relative to the peptide-free lipid systems due to penalties to maintain solvation of charged hydrophilic solutes within the membrane environment. PMID:25614183

Investigating Hydrophilic Pores in Model Lipid Bilayers Using Molecular Simulations: Correlating Bilayer Properties with Pore-Formation Thermodynamics.

PubMed

Hu, Yuan; Sinha, Sudipta Kumar; Patel, Sandeep

2015-06-23

Cell-penetrating and antimicrobial peptides show a remarkable ability to translocate across physiological membranes. Along with factors such as electric-potential-induced perturbations of membrane structure and surface tension effects, experiments invoke porelike membrane configurations during the solute transfer process into vesicles and cells. The initiation and formation of pores are associated with a nontrivial free-energy cost, thus necessitating a consideration of the factors associated with pore formation and the attendant free energies. Because of experimental and modeling challenges related to the long time scales of the translocation process, we use umbrella sampling molecular dynamics simulations with a lipid-density-based order parameter to investigate membrane-pore-formation free energy employing Martini coarse-grained models. We investigate structure and thermodynamic features of the pore in 18 lipids spanning a range of headgroups, charge states, acyl chain lengths, and saturation. We probe the dependence of pore-formation barriers on the area per lipid, lipid bilayer thickness, and membrane bending rigidities in three different lipid classes. The pore-formation free energy in pure bilayers and peptide translocating scenarios are significantly coupled with bilayer thickness. Thicker bilayers require more reversible work to create pores. The pore-formation free energy is higher in peptide-lipid systems than in peptide-free lipid systems due to penalties to maintain the solvation of charged hydrophilic solutes within the membrane environment.

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

PubMed Central

Kenworthy, Anne K.; Petranova, Nadezda; Edidin, Michael

2000-01-01

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface. PMID:10793141

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions.

PubMed

Sankhagowit, Shalene; Wu, Shao-Hua; Biswas, Roshni; Riche, Carson T; Povinelli, Michelle L; Malmstadt, Noah

2014-10-01

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles. Copyright © 2014 Elsevier B.V. All rights reserved.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

PubMed

Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

2017-08-22

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

Sub-millisecond ligand probing of cell receptors with multiple solution exchange

PubMed Central

Sylantyev, Sergiy; Rusakov, Dmitri A

2013-01-01

The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10–15 s. this protocol, which has been validated in our recent studies and takes 20–60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design. PMID:23744290

Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake.

PubMed

Chicca, Andrea; Nicolussi, Simon; Bartholomäus, Ruben; Blunder, Martina; Aparisi Rey, Alejandro; Petrucci, Vanessa; Reynoso-Moreno, Ines Del Carmen; Viveros-Paredes, Juan Manuel; Dalghi Gens, Marianela; Lutz, Beat; Schiöth, Helgi B; Soeberdt, Michael; Abels, Christoph; Charles, Roch-Philippe; Altmann, Karl-Heinz; Gertsch, Jürg

2017-06-20

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derived N -substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC 50 = 10 nM) inhibitor N -(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

Selectively Tuning a Buckled Si/SiO2 Membrane MEMS through Joule Heating Actuation and Mechanical Restriction

DTIC Science & Technology

2014-03-01

are solid substances whose electrical conductivity is between that of a conductor and that of an insulator, and are sometimes known as “poor...measurements were captured using a unique measurement scheme. The results concluded that by introducing a thermal stress, the membrane could be actuated with...61 Thermal Probe Station

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe.

PubMed

Ioffe, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

2007-06-01

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.

Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization.

PubMed

Wawrezinieck, Laure; Rigneault, Hervé; Marguet, Didier; Lenne, Pierre-François

2005-12-01

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.

Protons migrate along interfacial water without significant contributions from jumps between ionizable groups on the membrane surface

PubMed Central

Springer, Andreas; Hagen, Volker; Cherepanov, Dmitry A.; Antonenko, Yuri N.; Pohl, Peter

2011-01-01

Proton diffusion along membrane surfaces is thought to be essential for many cellular processes such as energy transduction. Commonly, it is treated as a succession of jumps between membrane-anchored proton-binding sites. Our experiments provide evidence for an alternative model. We released membrane-bound caged protons by UV flashes and monitored their arrival at distant sites by fluorescence measurements. The kinetics of the arrival is probed as a function of distance for different membranes and for different water isotopes. We found that proton diffusion along the membrane is fast even in the absence of ionizable groups in the membrane, and it decreases strongly in D2O as compared to H2O. We conclude that the fast proton transport along the membrane is dominated by diffusion via interfacial water, and not via ionizable lipid moieties. PMID:21859952

Peptide-coated semiconductor quantum dots and their applications in biological imaging of single molecules in live cells and organisms

NASA Astrophysics Data System (ADS)

Pinaud, Fabien Florent

2007-12-01

A new surface chemistry has been developed for the solubilization and biofunctionalization of inorganic semiconductor nanocrystals fluorescent probes, also known as quantum dots. This chemistry is based on the surface coating of quantum dots with custom-designed polycysteine peptides and yields water-soluble, small, monodispersed and colloidally stable probes that remain bright and photostable in complex biological milieus. This peptide coating strategy was successfully tested on several types of core and core-shell quantum dots emitting from the visible (e.g. CdSe/ZnS) to the NIR spectrum range (e.g. CdTe/CdSe/ZnS). By taking advantage of the versatile physico-chemical properties of peptides, a peptide "toolkit" was designed and employed to impart several biological functions to individual quantum dots and control their biochemical activity at the nanometer scale. These biofunctionalized peptide-coated quantum dots were exploited in very diverse biological applications. Near-infrared emitting quantum dot probes were engineered with optimized blood circulation and biodistribution properties for in vivo animal imaging. Visible emitting quantum dots were used for single molecule tracking of raft-associated GPI-anchored proteins in live cells. This last application revealed the presence of discrete and non-caveolar lipid microdomains capable of impeding free lateral diffusions in the plasma membrane of Hela cells. Imaging and tracking of peptide-coated quantum dots provided the first direct evidence that microdomains having the composition and behavior expected for lipid rafts can induce molecular compartmentalization in the membrane of living cells.

Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment.

PubMed

Horchani, Habib; de Saint-Jean, Maud; Barelli, Hélène; Antonny, Bruno

2014-01-01

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Beyond Fluorescent Proteins: Hybrid and Bioluminescent Indicators for Imaging Neural Activities.

PubMed

Wang, Anqi; Feng, Jiesi; Li, Yulong; Zou, Peng

2018-04-18

Optical biosensors have been invaluable tools in neuroscience research, as they provide the ability to directly visualize neural activity in real time, with high specificity, and with exceptional spatial and temporal resolution. Notably, a majority of these sensors are based on fluorescent protein scaffolds, which offer the ability to target specific cell types or even subcellular compartments. However, fluorescent proteins are intrinsically bulky tags, often insensitive to the environment, and always require excitation light illumination. To address these limitations, there has been a proliferation of alternative sensor scaffolds developed in recent years, including hybrid sensors that combine the advantages of synthetic fluorophores and genetically encoded protein tags, as well as bioluminescent probes. While still in their early stage of development as compared with fluorescent protein-based sensors, these novel probes have offered complementary solutions to interrogate various aspects of neuronal communication, including transmitter release, changes in membrane potential, and the production of second messengers. In this Review, we discuss these important new developments with a particular focus on design strategies.

A novel electrochemical biosensor based on dynamic polymerase-extending hybridization for E. coli O157:H7 DNA detection.

PubMed

Wang, Lijiang; Liu, Qingjun; Hu, Zhaoying; Zhang, Yuanfan; Wu, Chunsheng; Yang, Mo; Wang, Ping

2009-05-15

A novel biosensor based on single-stranded DNA (ssDNA) probe functionalized aluminum anodized oxide (AAO) nanopore membranes was demonstrated for Escherichia coli O157:H7 DNA detection. An original and dynamic polymerase-extending (PE) DNA hybridization procedure is proposed, where hybridization happens in the existence of Taq DNA polymerase and dNTPs under controlled reaction temperature. The probe strand would be extended as long as the target DNA strand, then the capability to block the ionic flow in the pores has been prominently enhanced by the double strand complex. We have investigated the variation of ionic conductivity during the fabrication of the film and the hybridization using cyclic voltammetry and impedance spectroscopy. The present approach provides low detection limit for DNA (a few hundreds of pmol), rapid label-free and easy-to-use bacteria detection, which holds the potential for future use in various ss-DNA analyses by integrated into a self-contained biochip.

Probing platinum degradation in polymer electrolyte membrane fuel cells by synchrotron X-ray microscopy.

PubMed

Berejnov, Viatcheslav; Martin, Zulima; West, Marcia; Kundu, Sumit; Bessarabov, Dmitri; Stumper, Jürgen; Susac, Darija; Hitchcock, Adam P

2012-04-14

Synchrotron-based scanning transmission X-ray spectromicroscopy (STXM) was used to characterize the local chemical environment at and around the platinum particles in the membrane (PTIM) which form in operationally tested (end-of-life, EOL) catalyst coated membranes (CCMs) of polymer electrolyte membrane fuel cells (PEM-FC). The band of metallic Pt particles in operationally tested CCM membranes was imaged using transmission electron microscopy (TEM). The cathode catalyst layer in the beginning-of-life (BOL) CCMs was fabricated using commercially available catalysts created from Pt precursors with and without nitrogen containing ligands. The surface composition of these catalyst powders was measured by X-ray Photoelectron Spectroscopy (XPS). The local chemical environment of the PTIM in EOL CCMs was found to be directly related to the Pt precursor used in CCM fabrication. STXM chemical mapping at the N 1s edge revealed a characteristic spectrum at and around the dendritic Pt particles in CCMs fabricated with nitrogen containing Pt-precursors. This N 1s spectrum was identical to that of the cathode and different from the membrane. For CCM samples fabricated without nitrogen containing Pt-precursors the N 1s spectrum at the Pt particles was indistinguishable from that of the adjacent membrane. We interpret these observations to indicate that nitrogenous ligands in the nitrogen containing precursors, or decomposition product(s) from that source, are transported together with the dissolved Pt from the cathode into the membrane as a result of the catalyst degradation process. This places constraints on possible mechanisms for the PTIM band formation process.

Liquid Droplets Act as "Compass Needles" for the Stresses in a Deformable Membrane.

PubMed

Schulman, Rafael D; Ledesma-Alonso, René; Salez, Thomas; Raphaël, Elie; Dalnoki-Veress, Kari

2017-05-12

We examine the shape of droplets atop deformable thin elastomeric films prepared with an anisotropic tension. As the droplets generate a deformation in the taut film through capillary forces, they assume a shape that is elongated along the high tension direction. By measuring the contact line profile, the tension in the membrane can be completely determined. Minimal theoretical arguments lead to predictions for the droplet shape and membrane deformation that are in excellent agreement with the data. On the whole, the results demonstrate that droplets can be used as probes to map out the stress field in a membrane.

The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles.

PubMed

Eklund, T

1985-01-01

The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated. Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential). Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.

Direct evidence of ionic fluxes across ion-selective membranes: a scanning electrochemical microscopic and potentiometric study.

PubMed

Gyurcsányi, R E; Pergel, E; Nagy, R; Kapui, I; Lan, B T; Tóth, K; Bitter, I; Lindner, E

2001-05-01

Scanning electrochemical microscopy (SECM) supplemented with potentiometric measurements was used to follow the time-dependent buildup of a steady-state diffusion layer at the aqueous-phase boundary of lead ion-selective electrodes (ISEs). Differential pulse voltammetry is adapted to SECM for probing the local concentration profiles at the sample side of solvent polymeric membranes. Major factors affecting the membrane transport-related surface concentrations were identified from SECM data and the potentiometric transients obtained under different experimental conditions (inner filling solution composition, membrane thickness, surface pretreatment). The amperometrically determined surface concentrations correlated well with the lower detection limits of the lead ion-selective electrodes.

Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based on an Apoferritin-Assembled Lanthanide Complex-Rhodamine Luminescence Resonance Energy Transfer System.

PubMed

Tian, Lu; Dai, Zhichao; Liu, Xiangli; Song, Bo; Ye, Zhiqiang; Yuan, Jingli

2015-11-03

Using apoferritin (AFt) as a carrier, a novel ratiometric luminescence probe based on luminescence resonance energy transfer (LRET) between a Tb(3+) complex (PTTA-Tb(3+)) and a rhodamine derivative (Rh-NO), PTTA-Tb(3+)@AFt-Rh-NO, has been designed and prepared for the specific recognition and time-gated luminescence detection of nitric oxide (NO) in living samples. In this LRET probe, PTTA-Tb(3+) encapsulated in the core of AFt is the energy donor, and Rh-NO, a NO-responsive rhodamine derivative, bound on the surface of AFt is the energy acceptor. The probe only emits strong Tb(3+) luminescence because the emission of rhodamine is switched off in the absence of NO. Upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from Tb(3+) complex to rhodamine occurs, which results in the remarkable increase and decrease of the long-lived emissions of rhodamine and PTTA-Tb(3+), respectively. After the reaction, the intensity ratio of rhodamine emission to Tb(3+) emission, I565/I539, is ∼24.5-fold increased, and the dose-dependent enhancement of I565/I539 shows a good linearity in a wide concentration range of NO. This unique luminescence response allowed PTTA-Tb(3+)@AFt-Rh-NO to be conveniently used as a ratiometric probe for the time-gated luminescence detection of NO with I565/I539 as a signal. Taking advantages of high specificity and sensitivity of the probe as well as its good water-solubility, biocompatibility, and cell membrane permeability, PTTA-Tb(3+)@AFt-Rh-NO was successfully used for the luminescent imaging of NO in living cells and Daphnia magna. The results demonstrated the efficacy of the probe and highlighted it's advantages for the ratiometric time-gated luminescence bioimaging application.

Changes Caused by Fruit Extracts in the Lipid Phase of Biological and Model Membranes

PubMed Central

Pruchnik, Hanna; Oszmiański, Jan; Sarapuk, Janusz; Kleszczyńska, Halina

2010-01-01

The aim of the study was to determine changes incurred by polyphenolic compounds from selected fruits in the lipid phase of the erythrocyte membrane, in liposomes formed of erythrocyte lipids and phosphatidylcholine liposomes. In particular, the effect of extracts from apple, chokeberry, and strawberry on the red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC liposomes was studied. In the erythrocyte population, the proportions of echinocytes increased due to incorporation of polyphenolic compounds. Fluorimetry with a laurdan probe indicated increased packing density in the hydrophilic phase of the membrane in presence of polyphenolic extracts, the highest effect being observed for the apple extract. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The polyphenolic extracts slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The studies have shown that the phenolic compounds contained in the extracts incorporate into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The compounds also penetrate the outer part of the external lipid layer of liposomes formed of natural and DPPC lipids, changing its packing order. PMID:21423329

Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

PubMed

Iwasaki, Fumihiko; Luginbühl, Sandra; Suga, Keishi; Walde, Peter; Umakoshi, Hiroshi

2017-02-28

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH 2 PO 4 ) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for intramembrane phase separations (coexistence of ordered and disordered domains). This means that highly fluid AOT membranes transform upon the addition of PADPA into membranes that have ordered domains. Although the relevance of this finding for the enzymatic oligomerization of PADPA is not yet clear, it is also of interest if one likes to use heterogeneous vesicle membranes as additives for carrying out membrane surface-confined reactions that do not necessarily involve PADPA as a reactant.

Specific binding of 15 HETE to lymphocytes. Effects on the fluidity of plasmatic membranes.

PubMed

Mexmain, S; Gualde, N; Aldigier, J C; Motta, C; Chable-Rabinovitch, H; Rigaud, M

1984-01-01

Specific binding of mouse lymphocytes for 15 HETE was examined by incubating cells with [14C]-15 HETE, 1 X 10(-8) to 1 X 10(-10)M. It was observed that the specific binding of radiolabeled 15 HETE is a function of time, of temperature and is modified by Ca2+ and dithiothreitol. When a fluorescent probe was embedded in the phospholipid core of the lymphocyte membrane and its motion analysed by fluorescence polarization, it was observed that 15 HETE increases the viscosity of the plasmatic membrane.

Diffusional dynamics of an active rhodamine-labeled 1,4-dihydropyridine in sarcolemmal lipid multibilayers.

PubMed Central

Mason, R P; Chester, D W

1989-01-01

A "membrane bilayer pathway" model, involving ligand partition into the bilayer, lateral diffusion, and receptor binding has been invoked to describe the 1,4-dihydropyridine (DHP) calcium channel antagonist receptor binding mechanism. In an earlier study (Chester et al. 1987. Biophys. J. 52:1021-1030), the diffusional component of this model was examined using an active fluorescence labeled DHP calcium channel antagonist, nisoldipine-lissamine rhodamine B (Ns-R), in purified cardiac sarcolemmal (CSL) lipid multibilayers. Diffusion coefficient measurements on membrane-bound drug and phospholipid at maximum bilayer hydration yielded similar values (3.8 x 10(-8) cm2/s). However, decreases in bilayer hydration resulted in dramatically reduced diffusion coefficient values for both probes with substantially greater impact on Ns-R diffusion. These data suggested that hydration dependent diffusional differences could be a function of relative probe location along the bilayer normal. In this communication, we have addressed the relative effect of the rhodamine substituent on Ns-R diffusion complex by examining the diffusional dynamics of free rhodamine B under the same conditions used to evaluate Ns-R complex and phospholipid diffusion. X-ray diffraction studies were performed to determine the Ns-R location in the membrane and model the CSL lipid bilayer profile structure to give a rationale for the differences in probe diffusional dynamics as a function of interbilayer water space. PMID:2611332

Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

PubMed

Huang, S K; Cheng, M; Hui, S W

1990-11-01

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.

In Vivo Imaging of Immuno-Spin Trapped Radicals With Molecular Magnetic Resonance Imaging in a Diabetic Mouse Model

PubMed Central

Towner, Rheal A.; Smith, Nataliya; Saunders, Debra; Henderson, Michael; Downum, Kristen; Lupu, Florea; Silasi-Mansat, Robert; Ramirez, Dario C.; Gomez-Mejiba, Sandra E.; Bonini, Marcelo G.; Ehrenshaft, Marilyn; Mason, Ronald P.

2012-01-01

Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes. PMID:22698922

Chemical Imaging of the Cell Membrane by NanoSIMS

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

Weber, P K; Kraft, M L; Frisz, J F

2010-02-23

The existence of lipid microdomains and their role in cell membrane organization are currently topics of great interest and controversy. The cell membrane is composed of a lipid bilayer with embedded proteins that can flow along the two-dimensional surface defined by the membrane. Microdomains, known as lipid rafts, are believed to play a central role in organizing this fluid system, enabling the cell membrane to carry out essential cellular processes, including protein recruitment and signal transduction. Lipid rafts are also implicated in cell invasion by pathogens, as in the case of the HIV. Therefore, understanding the role of lipid raftsmore » in cell membrane organization not only has broad scientific implications, but also has practical implications for medical therapies. One of the major limitations on lipid organization research has been the inability to directly analyze lipid composition without introducing artifacts and at the relevant length-scales of tens to hundreds of nanometers. Fluorescence microscopy is widely used due to its sensitivity and specificity to the labeled species, but only the labeled components can be observed, fluorophores can alter the behavior of the lipids they label, and the length scales relevant to imaging cell membrane domains are between that probed by fluorescence resonance energy transfer (FRET) imaging (<10 nm) and the diffraction limit of light. Topographical features can be imaged on this length scale by atomic force microscopy (AFM), but the chemical composition of the observed structures cannot be determined. Immuno-labeling can be used to study the distribution of membrane proteins at high resolution, but not lipid composition. We are using imaging mass spectrometry by secondary ion mass spectrometry (SIMS) in concert with other high resolution imaging methods to overcome these limitations. The experimental approach of this project is to combine molecule-specific stable isotope labeling with high-resolution SIMS using a Cameca NanoSIMS 50 to probe membrane organization and test microdomain hypotheses. The NanoSIMS is an imaging secondary ion mass spectrometer with an unprecedented combination of spatial resolution, sensitivity and mass specificity. It has 50 nm lateral resolution and is capable of detecting 1 in 20 nitrogen atoms while excluding near-neighbor isobaric interferences. The tightly focused cesium ion beam is rastered across the sample to produce simultaneous, quantitative digital images of up to five different masses. By labeling each specific components of a membrane with a unique rare stable isotope or element and mapping the location of the labels with the NanoSIMS, the location of the each labeled component can be determined and quantified. This new approach to membrane composition analysis allows molecular interactions of biological membranes to be probed at length-scales relevant to lipid rafts (10s to 100s of nm) that were not previously possible. Results from our most recent experiments analyzing whole cells will be presented.« less

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

PubMed

Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

2016-08-01

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, II: Fluorescence and Vibrational Spectroscopy Using a Cyanophenylalanine Probe

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

Liu, J.; Strzalka, J; Tronin, A

2009-01-01

We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-PheCN. The Trp to PheCN mutation alters neither the a-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this PheCN mutant hbAP-PheCN, based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescencemore » is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-PheCN, enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four PheCN probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-PheCN therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.« less

Hydration of sulfonated polyimide membranes. I. Na + and NH +(C 2H 5) 3 homopolymers

NASA Astrophysics Data System (ADS)

Jamróz, Dorota; Maréchal, Yves

2004-05-01

Hydration of Na + and HN +(C 2H 5) 3 sulfonated polyimide membranes is probed by infrared spectrometry using a recently described method. These membranes consist of identical sulfonated repeat unit (homopolymers) and form a good starting point to study more elaborated membranes, composed of these units plus similar ones with no sulfonic groups (block copolymers). These latter membranes may be used in fuel cells and will be described in a forthcoming article. We first identify the bands of hydrophilic groups, which will be anchor points for hydration. We then define three 'elementary hydration spectra' onto which all hydration spectra can be decomposed. Their analysis allows us to measure the water uptake of these membranes as a function of the hygrometry of the ambient atmosphere and to determine the structures of the dried membranes and their hydration mechanisms in terms of chemical reactions.

Detection and differentiation of Campylobacter jejuni and Campylobacter coli in broiler chicken samples using a PCR/DNA probe membrane based colorimetric detection assay.

PubMed

O'Sullivan, N A; Fallon, R; Carroll, C; Smith, T; Maher, M

2000-02-01

Campylobacter enteritis in humans has been linked to consumption of poultry meat. Surveys show that 30-100% of poultry harbour Campylobacter as normal flora of the digestive tract which indicates a need to identify prevalent organism types in flocks and trace their epidemiology. In this study we describe a Campylobacter genus specific polymerase chain reaction (PCR) assay, amplifying the 16 S-23 S rRNA intergenic spacer region with an internal Campylobacter genus specific DNA probe and species specific probes for Campylobacter jejuni and Campylobacter coli designed for confirmation of the amplified PCR products by Southern blot and colorimetric reverse hybridization assays. The specificity of this assay was established by testing a range of food pathogens. Broiler chicken samples were tested following presumptive positive identification by the Malthus System V analyser (Malthus Instruments, UK). The combined PCR and colorimetric reverse hybridization assay is easy to perform and faster than conventional methods for confirmation and identification of Campylobacter species. Copyright 2000 Academic Press.

Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone.

PubMed

Doucet, Christine M; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

2015-01-01

Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.

A novel method for determining calibration and behavior of PVDF ultrasonic hydrophone probes in the frequency range up to 100 MHz.

PubMed

Bleeker, H J; Lewin, P A

2000-01-01

A new calibration technique for PVDF ultrasonic hydrophone probes is described. Current implementation of the technique allows determination of hydrophone frequency response between 2 and 100 MHz and is based on the comparison of theoretically predicted and experimentally determined pressure-time waveforms produced by a focused, circular source. The simulation model was derived from the time domain algorithm that solves the non linear KZK (Khokhlov-Zabolotskaya-Kuznetsov) equation describing acoustic wave propagation. The calibration technique data were experimentally verified using independent calibration procedures in the frequency range from 2 to 40 MHz using a combined time delay spectrometry and reciprocity approach or calibration data provided by the National Physical Laboratory (NPL), UK. The results of verification indicated good agreement between the results obtained using KZK and the above-mentioned independent calibration techniques from 2 to 40 MHz, with the maximum discrepancy of 18% at 30 MHz. The frequency responses obtained using different hydrophone designs, including several membrane and needle probes, are presented, and it is shown that the technique developed provides a desirable tool for independent verification of primary calibration techniques such as those based on optical interferometry. Fundamental limitations of the presented calibration method are also examined.

A thiazolo[4,5-b]pyridine-based fluorescent probe for detection of zinc ions and application for in vitro and in vivo bioimaging.

PubMed

Gong, Jiao; Li, Yuan-Heng; Zhang, Chan-Juan; Huang, Jian; Sun, Qi

2018-08-01

2-HPTP, a novel thiazolo [4, 5-b] pyridine-based Zn 2+ selective fluorescent probe has been synthesized and investigated. This probe exhibited a high selectivity towards Zn 2+ over other biologically essential cations such as Na + , K + , Ca 2+ , or Mg 2+ . 2-HPTP formed a 1:1 complex with Zn 2+ and showed a fluorescent enhancement with a long emission wavelength red-shift (85 nm) upon complex formation with Zn 2+ . The detection limit and association constant were calculated as 3.48 × 10 -7 M and 2.40 × 10 6 M -1 by a fluorescence titration experiment. Furthermore, the live cell imaging experiment showed that 2-HPTP was membrane permeable and photostable, and hence, could be used to monitor the concentration changes of intracellular Zn 2+ . The co-staining experiments in the cells demonstrated that 2-HPTP possessed high lysosomal selectivity in living cells. Finally, using the nematode C. elegans as an experimental model, we established that 2-HPTP could be successful in imaging Zn 2+ concentration changes in living tissues. Therefore, this molecule should be useful for studies on the biological functions of Zn 2+ . Copyright © 2018 Elsevier B.V. All rights reserved.

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

PubMed Central

Naganathan, Saranga; Grunbeck, Amy; Tian, He; Huber, Thomas; Sakmar, Thomas P.

2013-01-01

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes. PMID:24056801

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

PubMed Central

Short, T. W.; Reymond, P.; Briggs, W. R.

1993-01-01

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase. PMID:12231721

Visual test of subparts per billion-level mercuric ion with a gold nanoparticle probe after preconcentration by hollow fiber supported liquid membrane.

PubMed

Tan, Zhi-qiang; Liu, Jing-fu

2010-05-15

With the combination of the gold nanoparticle (AuNP)-based visual test with hollow fiber supported liquid membrane (HFSLM) extraction, a highly sensitive and selective method was developed for field detection of mercuric ion (Hg(2+)) in environmental waters. Hg(2+) in water samples was extracted through HFSLM and trapped in the aqueous acceptor and then visually detected based on the red-to-blue color change of 3-mercaptopropionic acid-functionalized AuNP (MPA-AuNP) probe. The highest extraction efficiency of Hg(2+) was obtained by using a 600 mL sample (pH 8.0, 2.0% (w/v) NaCl), approximately 35 microL of acceptor (10 mM of 2,6-pyridinedicarboxylic acid, pH 4.0) filled in the lumen of a polypropylene hollow fiber tubing (55 cm in length, 50 microm wall thickness, 280 microm inner diameter), a liquid membrane of 2.0% (w/v) trioctycphosphine oxide in undecane, and a shaking rate of 250 rpm. The chromegenic reaction was conducted by incubating the mixture of MPA-AuNP stock solution (12 microL, 15 nM), Tris-borate buffer solution (18 microL, 0.2 M, pH 9.5), and acceptor (30 microL) at 30 degrees C for 1 h. The detection limit can be adjusted to 0.8 microg/L Hg(2+) (corresponding to an enrichment factor of approximately 1000 in the HFSLM) and 2.0 microg/L Hg(2+) (the U.S. Environmental Protection Agency limit of [Hg(2+)] for drinkable water) by using extraction times of 3 and 1 h, respectively. The proposed method is extremely specific for Hg(2+) with tolerance to at least 1000-fold of other environmentally relevant heavy and transition metal ions and was successfully applied to detect Hg(2+) in a certified reference water sample, as well as real river, lake, and tap water samples.

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy

PubMed Central

Dosoky, Noura Sayed; Patel, Darayas; Weimer, Jeffrey; Williams, John Dalton

2017-01-01

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature (Tm) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above—as well as below—the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10−2–105 Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm. The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures. PMID:28678160

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy.

PubMed

Khan, Muhammad Shuja; Dosoky, Noura Sayed; Patel, Darayas; Weimer, Jeffrey; Williams, John Dalton

2017-07-05

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature ( Tm ) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above-as well as below-the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10 -2 -10⁵ Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm . The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures.

Chemical Probes of Rapid Estrogen Signaling in Breast Cancer Treatment and Chemoprevention

DTIC Science & Technology

2007-04-01

The analogs will also be conju- gated to cell-impermeable polyacrylate polymers that should allow for selective targeting of membrane-initiated...the GW7604 analogs. Briefly, serial dilutions of the different compounds were prepared in ES2 screening buffer (100 mM potassium phosphate, pH7.4, 100...AD_________________ Award Number: W81XWH-04-1-0447 TITLE: CHEMICAL PROBES OF RAPID ESTROGEN

NMR Studies of Mass Transport in New Conducting Media for Fuel Cells

DTIC Science & Technology

2009-01-01

PEM films, for example those containing phosphoric acid and ionic liquids . Dynamical processes are probed at the short range by spin-lattice...structural environments of muticomponent PEM films, for example those containing phosphoric acid and ionic liquids . Dynamical processes are probed at the...correlation between water diffusivity and proton conductivity in the nanocomposites Transport properties of several ionic liquids (IL’s) and membranes

Development of Active DNA Control Technique for DNA Sequencer With a Solid-state Nanopore

NASA Astrophysics Data System (ADS)

Akahori, Rena; Harada, Kunio; Goto, Yusuke; Yanagi, Itaru; Yokoi, Takahide; Oura, Takeshi; Shibahara, Masashi; Takeda, Ken-Ichi

We have developed a technique that can control the arbitrary speeds of DNA passing through a solid-state nanopore of a DNA sequencer. For this active DNA control technique, we used a DNA-immobilized Si probe, larger than the membrane with a nanopore, and used a piezoelectric actuator and stepper motor to drive the probe. This probe enables a user to adjust the relative position between the nanopore and DNA immobilized on the probe without the need for precise lateral control. In this presentation, we demonstrate how DNA (block copolymer ([(dT)25-(dC)25-(dA)50]m)), immobilized on the probe, slid through a nanopore and was pulled out using the active DNA control technique. As the DNA-immobilized probe was being pulled out, we obtained various ion-current signal levels corresponding to the number of different nucleotides in a single strand of DNA.

Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes.

PubMed

Ohlsson, Gabriel; Tabaei, Seyed R; Beech, Jason; Kvassman, Jan; Johanson, Urban; Kjellbom, Per; Tegenfeldt, Jonas O; Höök, Fredrik

2012-11-21

Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.

Transmission Geometry Laser Ablation into a Non-Contact Liquid Vortex Capture Probe for Mass Spectrometry Imaging

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

Ovchinnikova, Olga S; Bhandari, Deepak; Lorenz, Matthias

2014-01-01

RATIONALE: Capture of material from a laser ablation plume into a continuous flow stream of solvent provides the means for uninterrupted sampling, transport and ionization of collected material for coupling with mass spectral analysis. Reported here is the use of vertically aligned transmission geometry laser ablation in combination with a new non-contact liquid vortex capture probe coupled with electrospray ionization for spot sampling and chemical imaging with mass spectrometry. Methods: A vertically aligned continuous flow liquid vortex capture probe was positioned directly underneath a sample surface in a transmission geometry laser ablation (355 nm, 10 Hz, 7 ns pulse width)more » setup to capture into solution the ablated material. The outlet of the vortex probe was coupled to the Turbo V ion source of an AB SCIEX TripleTOF 5600+ mass spectrometer. System operation and performance metrics were tested using inked patterns and thin tissue sections. Glass slides and slides designed especially for laser capture microdissection, viz., DIRECTOR slides and PEN 1.0 (polyethylene naphthalate) membrane slides, were used as sample substrates. Results: The estimated capture efficiency of laser ablated material was 24%, which was enabled by the use of a probe with large liquid surface area (~ 2.8 mm2) and with gravity to help direct ablated material vertically down towards the probe. The swirling vortex action of the liquid surface potentially enhanced capture and dissolution of not only particulates, but also gaseous products of the laser ablation. The use of DIRECTOR slides and PEN 1.0 (polyethylene naphthalate) membrane slides as sample substrates enabled effective ablation of a wide range of sample types (basic blue 7, polypropylene glycol, insulin and cyctochrome c) without photodamage using a UV laser. Imaging resolution of about 6 m was demonstrated for stamped ink on DIRECTOR slides based on the ability to distinguish features present both in the optical and in the chemical image. This imaging resolution was 20 times better than the previous best reported results with laser ablation/liquid sample capture mass spectrometry imaging. Using thin sections of brain tissue the chemical image of a selected lipid was obtained with an estimated imaging resolution of about 50 um. Conclusions: A vertically aligned, transmission geometry laser ablation liquid vortex capture probe, electrospray ionization mass spectrometry system provides an effective means for spatially resolved spot sampling and imaging with mass spectrometry.« less

Transmission geometry laser ablation into a non-contact liquid vortex capture probe for mass spectrometry imaging.

PubMed

Ovchinnikova, Olga S; Bhandari, Deepak; Lorenz, Matthias; Van Berkel, Gary J

2014-08-15

Capture of material from a laser ablation plume into a continuous flow stream of solvent provides the means for uninterrupted sampling, transport and ionization of collected material for coupling with mass spectral analysis. Reported here is the use of vertically aligned transmission geometry laser ablation in combination with a new non-contact liquid vortex capture probe coupled with electrospray ionization for spot sampling and chemical imaging with mass spectrometry. A vertically aligned continuous flow liquid vortex capture probe was positioned directly underneath a sample surface in a transmission geometry laser ablation (355 nm, 10 Hz, 7 ns pulse width) set up to capture into solution the ablated material. The outlet of the vortex probe was coupled to the Turbo V™ ion source of an AB SCIEX TripleTOF 5600+ mass spectrometer. System operation and performance metrics were tested using inked patterns and thin tissue sections. Glass slides and slides designed especially for laser capture microdissection, viz., DIRECTOR(®) slides and PEN 1.0 (polyethylene naphthalate) membrane slides, were used as sample substrates. The estimated capture efficiency of laser-ablated material was 24%, which was enabled by the use of a probe with large liquid surface area (~2.8 mm(2) ) and with gravity to help direct ablated material vertically down towards the probe. The swirling vortex action of the liquid surface potentially enhanced capture and dissolution not only of particulates, but also of gaseous products of the laser ablation. The use of DIRECTOR(®) slides and PEN 1.0 (polyethylene naphthalate) membrane slides as sample substrates enabled effective ablation of a wide range of sample types (basic blue 7, polypropylene glycol, insulin and cyctochrome c) without photodamage using a UV laser. Imaging resolution of about 6 µm was demonstrated for stamped ink on DIRECTOR(®) slides based on the ability to distinguish features present both in the optical and in the chemical image. This imaging resolution was 20 times better than the previous best reported results with laser ablation/liquid sample capture mass spectrometry imaging. Using thin sections of brain tissue the chemical image of a selected lipid was obtained with an estimated imaging resolution of about 50 µm. A vertically aligned, transmission geometry laser ablation liquid vortex capture probe, electrospray ionization mass spectrometry system provides an effective means for spatially resolved spot sampling and imaging with mass spectrometry. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

PubMed

Sahu, Indra D; Zhang, Rongfu; Dunagan, Megan M; Craig, Andrew F; Lorigan, Gary A

2017-06-01

EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (T m ) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the T m titration experiment indicated an increase in T m values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant differences in EPR spectral line broadening and a corresponding inverse central line width between spin-labeled KCNE1 residues located inside and outside of the membrane for lipodisq nanoparticle samples when compared to lipid vesicle samples. These results are consistent with the solution NMR structure of KCNE1. This study will be beneficial for researchers working on studying the structural and dynamic properties of membrane proteins.

The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

PubMed Central

Miller, Evan W.; Slak Rupnik, Marjan

2013-01-01

Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. PMID:24324777

Induction of Intracellular Reductive Stress with a Photoactivatable Phosphine Probe.

PubMed

Tirla, Alina; Rivera-Fuentes, Pablo

2018-04-25

Reductive stress is a condition present in cells that have an increased concentration of reducing species, and it has been associated with a number of pathologies, such as neurodegenerative diseases and cancer. The tools available to study reductive stress lack both in selectivity and specific targeting and some of these shortcomings can be addressed by using photoactivatable compounds. We developed a photoactivatable phosphonium probe, which upon irradiation releases a fluorescent molecule and a trialkyphosphine. The probes can permeate through the plasma membrane and the photoreleased phosphine can induce intracellular reductive stress as proven by the detection of protein aggregates.

Two-Photon Fluorescent Probe for Monitoring Autophagy via Fluorescence Lifetime Imaging.

PubMed

Hou, Liling; Ning, Peng; Feng, Yan; Ding, Yaqi; Bai, Lei; Li, Lin; Yu, Haizhu; Meng, Xiangming

2018-06-19

We reported the first lysosome targeted two-photon fluorescent probe (Lyso-NP) as a viscosity probe for monitoring autophagy. The fluorescence lifetime of Lyso-NP exhibited an excellent linear relationship with viscosity value ( R 2 = 0.99, x = 0.39). Lyso-NP also showed the specific capability for imaging lysosomal viscosity under two-photon excitation at 860 nm along with good biocompatibility. More importantly, Lyso-NP could be used to monitor the autophagy process in living cells by quantitatively detecting lysosomal viscosity changes during the membrane fusion process via two-photon fluorescence lifetime imaging.

Laser damage of free-standing nanometer membranes

NASA Astrophysics Data System (ADS)

Morimoto, Yuya; Roland, Iännis; Rennesson, Stéphanie; Semond, Fabrice; Boucaud, Philippe; Baum, Peter

2017-12-01

Many high-field/attosecond and ultrafast electron diffraction/microscopy experiments on condensed matter require samples in the form of free-standing membranes with nanometer thickness. Here, we report the measurement of the laser-induced damage threshold of 11 different free-standing nanometer-thin membranes of metallic, semiconducting, and insulating materials for 1-ps, 1030-nm laser pulses at 50 kHz repetition rate. We find a laser damage threshold that is very similar to each corresponding bulk material. The measurements also reveal a band gap dependence of the damage threshold as a consequence of different ionization rates. These results establish the suitability of free-standing nanometer membranes for high-field pump-probe experiments.

Use of the pH sensitive fluorescence probe pyranine to monitor internal pH changes in Escherichia coli membrane vesicles.

PubMed

Damiano, E; Bassilana, M; Rigaud, J L; Leblanc, G

1984-01-23

Measurements of the fluorescent properties of 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) enclosed within the internal space of Escherichia coli membrane vesicles enable recordings and quantitative analysis of: (i) changes in intravesicular pH taking place during oxidation of electron donors by the membrane respiratory chain; (ii) transient alkalization of the internal aqueous space resulting from the creation of outwardly directed acetate diffusion gradients across the vesicular membrane. Quantitation of the fluorescence variations recorded during the creation of transmembrane acetate gradients shows a close correspondence between the measured shifts in internal pH value and those expected from the amplitude of the imposed acetate gradients.

Usefulness of 1000-Hz probe tone in tympanometry according to age in Korean infants.

PubMed

Park, Mina; Han, Kyu-Hee; Jung, Hyunseo; Kim, Mee-Hee; Chang, Hyun-Kyung; Kim, Shin Hye; Park, Moo Kyun; Lee, Jun Ho

2015-01-01

Numerous studies have shown the superiority of a 1000-Hz frequency probe tone for evaluating the middle ear status of infants. However, most of these studies examined Caucasian populations. This study validated the 1000-Hz probe tone and evaluated the age at which it should be used in Korean infants. Data from 83 infants (43 males, 40 females; mean age 9.2±6.2 (range 1-30) months, 165 ears) were analyzed. Tympanograms were classified according to Baldwin's modification of the method of Marchant et al. and correlated with results based on combined diagnostic tests, including an endoscopic examination of the tympanic membrane, myringotomy findings, and the air and bone conduction auditory brainstem response (ABR) thresholds. Data were analyzed in five age groups, each covering a 3-month range. The traces were measured for both 226- and 1000-Hz probe tones. The sensitivity and specificity for the different age groups were also determined. For the 226-Hz probe tone, the tympanograms showed normal traces for most ears with otitis media effusions in infants younger than 12 months. By contrast, the tympanograms using the 1000-Hz probe tone showed abnormal traces in most of the infants with otitis media effusions in all age groups. In infants with no otitis media effusion, the tympanograms using both 226- and 1000-Hz probe tones were interpreted as normal in most cases in all age groups. In infants younger than 12 months, the sensitivity of the 226-Hz probe tone was very low (0-6.6%), whereas that of the 1000-Hz probe tone was very high (90-100%). In infants older than 13 months, however, the sensitivities of the 226- and 1000-Hz probe tones were 76.2% and 85.7%, respectively. Regarding specificity, the difference between the two probe tones was not significant for any age group. This study confirmed the superiority of the 1000-Hz probe tone for evaluating the middle ear in infants. We recommend using a 1000-Hz probe tone at least up to the age of 12 months for Korean infants. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Electronic waste leachate-mediated DNA fragmentation and cell death by apoptosis in mouse fibroblast (NIH/3T3) cell line.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Filippin-Monteiro, Fabíola B; Sierra, Jelver A; Creczynski-Pasa, Tânia B

2013-08-01

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N-acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N-acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

A Modified Alderman-Grant Coil makes possible an efficient cross-coil probe for high field solid-state NMR of lossy biological samples

NASA Astrophysics Data System (ADS)

Grant, Christopher V.; Yang, Yuan; Glibowicka, Mira; Wu, Chin H.; Park, Sang Ho; Deber, Charles M.; Opella, Stanley J.

2009-11-01

The design, construction, and performance of a cross-coil double-resonance probe for solid-state NMR experiments on lossy biological samples at high magnetic fields are described. The outer coil is a Modified Alderman-Grant Coil (MAGC) tuned to the 1H frequency. The inner coil consists of a multi-turn solenoid coil that produces a B 1 field orthogonal to that of the outer coil. This results in a compact nested cross-coil pair with the inner solenoid coil tuned to the low frequency detection channel. This design has several advantages over multiple-tuned solenoid coil probes, since RF heating from the 1H channel is substantially reduced, it can be tuned for samples with a wide range of dielectric constants, and the simplified circuit design and high inductance inner coil provides excellent sensitivity. The utility of this probe is demonstrated on two electrically lossy samples of membrane proteins in phospholipid bilayers (bicelles) that are particularly difficult for conventional NMR probes. The 72-residue polypeptide embedding the transmembrane helices 3 and 4 of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (residues 194-241) requires a high salt concentration in order to be successfully reconstituted in phospholipid bicelles. A second application is to paramagnetic relaxation enhancement applied to the membrane-bound form of Pf1 coat protein in phospholipid bicelles where the resistance to sample heating enables high duty cycle solid-state NMR experiments to be performed.

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample.

PubMed

Ootsubo, M; Shimizu, T; Tanaka, R; Sawabe, T; Tajima, K; Ezura, Y

2003-01-01

A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers*

PubMed Central

Antonenko, Yuri N.; Avetisyan, Armine V.; Cherepanov, Dmitry A.; Knorre, Dmitry A.; Korshunova, Galina A.; Markova, Olga V.; Ojovan, Silvia M.; Perevoshchikova, Irina V.; Pustovidko, Antonina V.; Rokitskaya, Tatyana I.; Severina, Inna I.; Simonyan, Ruben A.; Smirnova, Ekaterina A.; Sobko, Alexander A.; Sumbatyan, Natalia V.; Severin, Fedor F.; Skulachev, Vladimir P.

2011-01-01

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease. PMID:21454507

Proton conductivity and methanol permeability of Nafion-SiO2/SiWA composite membranes

NASA Astrophysics Data System (ADS)

Thiam, Hui San; Chia, Min Yan; Cheah, Qiao Rou; Koo, Charlene Chai Hoon; Lai, Soon Onn; Chong, Kok Chung

2017-04-01

Proton exchange membranes for a direct methanol fuel cell (DMFC) were prepared by incorporating silica/silicotungstic acid (SiO2/SiWA) inorganic composite into a Nafion polymer. The effects of SiO2/SiWA content on proton conductivity of membranes were investigated by using a four-probe conductivity cell. Methanol permeability of composite membrane was also determined by using a homemade diffusion cell and gas chromatography technique. It was found that proton conductivity of the composite membranes decreased with SiO2/SiWA content, however the highest proton conductivity achieved was 11% greater than the pure recast Nafion membrane. The methanol permeability of composite membrane was much lower than that of pure recast Nafion, in a reduction of 58% which indicated a better resistance to fuel crossover. Nafion-SiO2/SiWA composite membrane showed promising advantages over pure Nafion on electrochemical properties such as proton conductivity and fuel crossover and it is potentially attractive for use in DMFC.

The Sigma-2 (σ2) Receptor: A Novel Protein for the Imaging and Treatment of Cancer

PubMed Central

Mach, Robert H.; Zeng, Chenbo; Hawkins, William G.

2014-01-01

The sigma-2 (σ2) receptor is an important target for the development of molecular probes in oncology because of its 10-fold higher density in proliferating tumor cells than in quiescent tumor cells, and the observation that σ2 receptor agonists are able to kill tumor cells via apoptotic and non-apoptotic mechanisms. Although recent evidence indicates the σ2 receptor binding site is localized within the progesterone receptor membrane component 1 (PGRMC1), most information regarding this protein has been obtained using either radiolabeled or fluorescent receptor-based probes, and from biochemical analysis of the effect of σ2 selective ligands on cells grown in culture. This article reviews the development of σ2 receptor ligands, and presents an overview of how they have been used in vitro and in vivo to increase our understanding of the role of the σ2 receptor in cancer and proliferation. PMID:23734634

Fracture Tests of Etched Components Using a Focused Ion Beam Machine

NASA Technical Reports Server (NTRS)

Kuhn, Jonathan, L.; Fettig, Rainer K.; Moseley, S. Harvey; Kutyrev, Alexander S.; Orloff, Jon; Powers, Edward I. (Technical Monitor)

2000-01-01

Many optical MEMS device designs involve large arrays of thin (0.5 to 1 micron components subjected to high stresses due to cyclic loading. These devices are fabricated from a variety of materials, and the properties strongly depend on size and processing. Our objective is to develop standard and convenient test methods that can be used to measure the properties of large numbers of witness samples, for every device we build. In this work we explore a variety of fracture test configurations for 0.5 micron thick silicon nitride membranes machined using the Reactive Ion Etching (RIE) process. Testing was completed using an FEI 620 dual focused ion beam milling machine. Static loads were applied using a probe. and dynamic loads were applied through a piezo-electric stack mounted at the base of the probe. Results from the tests are presented and compared, and application for predicting fracture probability of large arrays of devices are considered.

Role of gemfibrozil as an inhibitor of CYP2C8 and membrane transporters.

PubMed

Tornio, Aleksi; Neuvonen, Pertti J; Niemi, Mikko; Backman, Janne T

2017-01-01

Cytochrome P450 (CYP) 2C8 is a drug metabolizing enzyme of major importance. The lipid-lowering drug gemfibrozil has been identified as a strong inhibitor of CYP2C8 in vivo. This effect is due to mechanism-based inhibition of CYP2C8 by gemfibrozil 1-O-β-glucuronide. In vivo, gemfibrozil is a fairly selective CYP2C8 inhibitor, which lacks significant inhibitory effect on other CYP enzymes. Gemfibrozil can, however, have a smaller but clinically meaningful inhibitory effect on membrane transporters, such as organic anion transporting polypeptide 1B1 and organic anion transporter 3. Areas covered: This review describes the inhibitory effects of gemfibrozil on CYP enzymes and membrane transporters. The clinical drug interactions caused by gemfibrozil and the different mechanisms contributing to the interactions are reviewed in detail. Expert opinion: Gemfibrozil is a useful probe inhibitor of CYP2C8 in vivo, but its effect on membrane transporters has to be taken into account in study design and interpretation. Moreover, gemfibrozil could be used to boost the pharmacokinetics of CYP2C8 substrate drugs. Identification of gemfibrozil 1-O-β-glucuronide as a potent mechanism-based inhibitor of CYP2C8 has led to recognition of glucuronide metabolites as perpetrators of drug-drug interactions. Recently, also acyl glucuronide metabolites of clopidogrel and deleobuvir have been shown to strongly inhibit CYP2C8.

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid.

PubMed

Tokunaga, Tomohisa; Watanabe, Bunta; Sato, Sho; Kawamoto, Jun; Kurihara, Tatsuo

2017-08-16

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C-C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to cis double bonds. One double bond and an ω-terminal C-C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, Shewanella livingstonensis Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA.

Using confocal laser scanning microscopy to probe the milk fat globule membrane and associated proteins.

PubMed

Gallier, Sophie; Gragson, Derek; Jiménez-Flores, Rafael; Everett, David

2010-04-14

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Direct measurement of lateral transport in membranes by using time-resolved spatial photometry.

PubMed Central

Kapitza, H G; McGregor, G; Jacobson, K A

1985-01-01

Spatially resolving light detectors allow, with proper calibration, quantitative analysis of the variations in two-dimensional intensity distributions over time. An ultrasensitive microfluorometer was assembled by using as a detector a microchannel plate-intensified video camera. The camera was interfaced with a software-based digital video analysis system to digitize, average, and process images and to directly control the timing of the experiments to minimize exposure of the specimen to light. The detector system has been characterized to allow its use as a photometer. A major application has been to perform fluorescence recovery after photobleaching measurements by using the camera in place of a photomultiplier tube (video-FRAP) with the goal of detecting possible anisotropic diffusion or convective flow. Analysis of the data on macromolecular diffusion in homogenous aqueous glycol solutions yielded diffusion constants in agreement with previous measurements. Results on lipid probe diffusion in dimyristoylphosphatidylcholine multibilayers indicated that at temperatures above the gel-to-liquid crystalline phase transition diffusion is isotropic, and analysis of video-FRAP data yielded diffusion coefficients consistent with those measured previously by using spot photobleaching. However, lipid probes in these multibilayers held just below the main phase transition temperature exhibited markedly anisotropic diffusive fluxes when the bleaching beam was positioned proximate to domain boundaries in the P beta' phase. Lipid probes and lectin receptor complexes diffused isotropically in fibroblast surface membranes with little evidence for diffusion channeled parallel to stress fibers. A second application was to trace the time evolution of cell surface reactions such as patching. The feasibility of following, on the optical scale, the growth of individual receptor clusters induced by the ligand wheat germ agglutinin was demonstrated. PMID:3858869

Direct measurement of lateral transport in membranes by using time-resolved spatial photometry.

PubMed

Kapitza, H G; McGregor, G; Jacobson, K A

1985-06-01

Spatially resolving light detectors allow, with proper calibration, quantitative analysis of the variations in two-dimensional intensity distributions over time. An ultrasensitive microfluorometer was assembled by using as a detector a microchannel plate-intensified video camera. The camera was interfaced with a software-based digital video analysis system to digitize, average, and process images and to directly control the timing of the experiments to minimize exposure of the specimen to light. The detector system has been characterized to allow its use as a photometer. A major application has been to perform fluorescence recovery after photobleaching measurements by using the camera in place of a photomultiplier tube (video-FRAP) with the goal of detecting possible anisotropic diffusion or convective flow. Analysis of the data on macromolecular diffusion in homogenous aqueous glycol solutions yielded diffusion constants in agreement with previous measurements. Results on lipid probe diffusion in dimyristoylphosphatidylcholine multibilayers indicated that at temperatures above the gel-to-liquid crystalline phase transition diffusion is isotropic, and analysis of video-FRAP data yielded diffusion coefficients consistent with those measured previously by using spot photobleaching. However, lipid probes in these multibilayers held just below the main phase transition temperature exhibited markedly anisotropic diffusive fluxes when the bleaching beam was positioned proximate to domain boundaries in the P beta' phase. Lipid probes and lectin receptor complexes diffused isotropically in fibroblast surface membranes with little evidence for diffusion channeled parallel to stress fibers. A second application was to trace the time evolution of cell surface reactions such as patching. The feasibility of following, on the optical scale, the growth of individual receptor clusters induced by the ligand wheat germ agglutinin was demonstrated.

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry

PubMed Central

Wilson, Robert L.; Frisz, Jessica F.; Hanafin, William P.; Carpenter, Kevin J.; Hutcheon, Ian D.; Weber, Peter K.; Kraft, Mary L.

2014-01-01

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein’s activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests of whether specific proteins co-localize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein co-localization with specific lipid species. PMID:22284327

Lipid peroxidation in sarcoplasmic reticulum membranes: effect on functional and biophysical properties.

PubMed

Dinis, T C; Almeida, L M; Madeira, V M

1993-03-01

The fluorescent polyunsaturated parinaric acid (PnA) incorporated in sarcoplasmic reticulum membranes (SR) was used to probe the initial stages of membrane lipid peroxidation. The experimental set up of the PnA assay was investigated by means of several peroxidation initiators to ascertain peroxidation conditions. This assay in SR is particularly useful to evaluate the membrane susceptibility to peroxidation and to ascertain suitable conditions (concentration of initiators and cofactors) to challenge peroxidation in each preparation under study. On the basis of the PnA assay, Fe2+/ascorbate was selected among the different initiator systems to assess the effect of lipid peroxidation upon biochemical and biophysical parameters of SR membranes. Under mildly controlled conditions at 25 degrees C, the lipid degradative process, as detected by fatty acid analysis, decreases the Ca2+ uptake (up to about 50% of control) and reduces the Ca2+ pump efficiency (Ca2+/ATP ratio) up to about 58% of control, without inactivation the ATPase enzyme turnover. The effect of lipid peroxidation on the SR bilayer organization is dependent either on the extent of lipid peroxidation or on the depth of the bilayer as probed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and by intramolecular excimerization of 1,3-di(1-pyrenyl)propane. It is concluded that the effect of mild lipid peroxidation on Ca2+ pump activity is partially exerted through the alteration of physical properties in the lipid phase or lipid-protein interfaces.

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.

Hematoporphyrin derivative induced photodamage to brain tumor cells: Alterations in subcellular membranes

NASA Astrophysics Data System (ADS)

Sreenivasan, Rajesh; Joshi, Preeti G.; Joshi, Nanda B.

1997-01-01

Photoinduced structural and functional changes were studied in the subcellular membranes isolated from HpD treated cells. Changes in the limiting anisotropy of lipid specific probes 1,6,Diphenyl-1,3,5,hexatriene (DPH) and 1-(4-Trimethyl ammonium 1,6 diphenyl)-1,3,5,hexatriene toulene sulphonate (TMA-DPH) incorporated into the membrane were used to assess the structural alterations while changes in the activity of the marker enzymes were used to assess the functional alterations. Our results suggest that damage to the endoplasmic reticulum may play an important role in the photosensitization of brain tumor cells.

A novel squarylium dye for monitoring oxidative processes in lipid membranes.

PubMed

Trusova, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

2009-11-01

A novel squaraine probe SQ-1 has been found to be appropriate for monitoring the peroxidation processes in membrane systems. Formation of free radicals was triggered by methemoglobin (metHb) or cytochrome c (cyt c) binding to the model lipid membranes composed of zwitterionic lipid phosphatidylcholine (PC) and anionic lipid cardiolipin (CL). Protein association with the lipid vesicles was followed by drastic quenching of SQ-1 fluorescence. The observed spectral changes were suppressed in the presence of free radical scavengers, butylated hydroxytoluene (BHT) and thiourea (TM) suggesting that SQ-1 decolorization can be attributed to its reactions with lipid radicals.

Chemical Probes of Rapid Estrogen Signaling in Breast Cancer Treatment and Chemoprevention

DTIC Science & Technology

2006-04-01

conjugated to cell-impermeable polyacrylate polymers that should allow for selective targeting of membrane-initiated responses of estrogen receptor. It...dilutions of the different compounds were prepared in ES2 screening buffer (100 mM potassium phosphate, pH7.4, 100 µg/ml bovine gamma globulin) and 50 µl...W81XWH-04-1-0447 TITLE: Chemical Probes of Rapid Estrogen Signaling in Breast Cancer Treatment and Chemoprevention PRINCIPAL

Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update.

PubMed

Wolff, Antonio C; Hammond, M Elizabeth Hale; Allison, Kimberly H; Harvey, Brittany E; Mangu, Pamela B; Bartlett, John M S; Bilous, Michael; Ellis, Ian O; Fitzgibbons, Patrick; Hanna, Wedad; Jenkins, Robert B; Press, Michael F; Spears, Patricia A; Vance, Gail H; Viale, Giuseppe; McShane, Lisa M; Dowsett, Mitchell

2018-05-30

- To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. - Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. - Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in >10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended workup for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥2.0; average HER2 copy number <4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥4.0 and <6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results.

Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics.

PubMed

Pazos, Ileana M; Ahmed, Ismail A; Berríos, Mariana I León; Gai, Feng

2015-08-15

We expand the spectroscopic utility of a well-known infrared and fluorescence probe, p-cyanophenylalanine, by showing that it can also serve as a pH sensor. This new application is based on the notion that the fluorescence quantum yield of this unnatural amino acid, when placed at or near the N-terminal end of a polypeptide, depends on the protonation status of the N-terminal amino group of the peptide. Using this pH sensor, we are able to determine the N-terminal pKa values of nine tripeptides and also the membrane penetration kinetics of a cell-penetrating peptide. Taken together, these examples demonstrate the applicability of using this unnatural amino acid fluorophore to study pH-dependent biological processes or events that accompany a pH change. Copyright © 2015 Elsevier Inc. All rights reserved.

Online determination of biophysical parameters of mucous membranes of a human body

NASA Astrophysics Data System (ADS)

Lisenko, S. A.; Kugeiko, M. M.

2013-07-01

We have developed a method for online determination of biophysical parameters of mucous membranes (MMs) of a human body (transport scattering coefficient, scattering anisotropy factor, haemoglobin concentration, degrees of blood oxygenation, average diameter of capillaries with blood) from measurements of spectral and spatial characteristics of diffuse reflection. The method is based on regression relationships between linearly independent components of the measured light signals and the unknown parameters of MMs, obtained by simulation of the radiation transfer in the MM under conditions of its general variability. We have proposed and justified the calibration-free fibre-optic method for determining the concentration of haemoglobin in MMs by measuring the light signals diffusely reflected by the tissue in four spectral regions at two different distances from the illumination spot. We have selected the optimal wavelengths of optical probing for the implementation of the method.

A potential target for organophosphate insecticides leading to spermatotoxicity.

PubMed

Suzuki, Himiko; Tomizawa, Motohiro; Ito, Yuki; Abe, Keisuke; Noro, Yuki; Kamijima, Michihiro

2013-10-16

Organophosphate (OP) insecticides as an anticholinesterase also act on the diverse serine hydrolase targets, thereby revealing secondary or unexpected toxic effects including male reproductive toxicity. The present investigation detects a possible target molecule(s) for OP-induced spermatotoxicity (sperm deformity, underdevelopment, and reduced motility) from a chemical standpoint. The activity-based protein profiling (ABPP) approach with a phosphonofluoridate fluorescent probe pinpointed the molecular target for fenitrothion (FNT, a major OP insecticide) oxon (bioactive metabolite of FNT) in the mouse testicular membrane proteome, i.e., FNT oxon phosphorylates the fatty acid amide hydrolase (FAAH), which plays pivotal roles in spermatogenesis and sperm motility acquirement. Subsequently, mice were treated orally with vehicle or FNT for 10 days, and FAAH activity in testis or epididymis cauda was markedly reduced by the subacute exposure. ABPP analysis revealed that FAAH was selectively inhibited among the FNT-treated testicular membrane proteome. Accordingly, FAAH is a potential target for OP-elicited spermatotoxicity.

Simultaneous multi-patch-clamp and extracellular-array recordings: Single neuron reflects network activity

NASA Astrophysics Data System (ADS)

Vardi, Roni; Goldental, Amir; Sardi, Shira; Sheinin, Anton; Kanter, Ido

2016-11-01

The increasing number of recording electrodes enhances the capability of capturing the network’s cooperative activity, however, using too many monitors might alter the properties of the measured neural network and induce noise. Using a technique that merges simultaneous multi-patch-clamp and multi-electrode array recordings of neural networks in-vitro, we show that the membrane potential of a single neuron is a reliable and super-sensitive probe for monitoring such cooperative activities and their detailed rhythms. Specifically, the membrane potential and the spiking activity of a single neuron are either highly correlated or highly anti-correlated with the time-dependent macroscopic activity of the entire network. This surprising observation also sheds light on the cooperative origin of neuronal burst in cultured networks. Our findings present an alternative flexible approach to the technique based on a massive tiling of networks by large-scale arrays of electrodes to monitor their activity.

Simultaneous multi-patch-clamp and extracellular-array recordings: Single neuron reflects network activity.

PubMed

Vardi, Roni; Goldental, Amir; Sardi, Shira; Sheinin, Anton; Kanter, Ido

2016-11-08

The increasing number of recording electrodes enhances the capability of capturing the network's cooperative activity, however, using too many monitors might alter the properties of the measured neural network and induce noise. Using a technique that merges simultaneous multi-patch-clamp and multi-electrode array recordings of neural networks in-vitro, we show that the membrane potential of a single neuron is a reliable and super-sensitive probe for monitoring such cooperative activities and their detailed rhythms. Specifically, the membrane potential and the spiking activity of a single neuron are either highly correlated or highly anti-correlated with the time-dependent macroscopic activity of the entire network. This surprising observation also sheds light on the cooperative origin of neuronal burst in cultured networks. Our findings present an alternative flexible approach to the technique based on a massive tiling of networks by large-scale arrays of electrodes to monitor their activity.

Simultaneous multi-patch-clamp and extracellular-array recordings: Single neuron reflects network activity

PubMed Central

Vardi, Roni; Goldental, Amir; Sardi, Shira; Sheinin, Anton; Kanter, Ido

2016-01-01

The increasing number of recording electrodes enhances the capability of capturing the network’s cooperative activity, however, using too many monitors might alter the properties of the measured neural network and induce noise. Using a technique that merges simultaneous multi-patch-clamp and multi-electrode array recordings of neural networks in-vitro, we show that the membrane potential of a single neuron is a reliable and super-sensitive probe for monitoring such cooperative activities and their detailed rhythms. Specifically, the membrane potential and the spiking activity of a single neuron are either highly correlated or highly anti-correlated with the time-dependent macroscopic activity of the entire network. This surprising observation also sheds light on the cooperative origin of neuronal burst in cultured networks. Our findings present an alternative flexible approach to the technique based on a massive tiling of networks by large-scale arrays of electrodes to monitor their activity. PMID:27824075

Quasi-optical antenna-mixer-array design for terahertz frequencies

NASA Technical Reports Server (NTRS)

Guo, Yong; Potter, Kent A.; Rutledge, David B.

1992-01-01

A new quasi-optical antenna-mixer-array design for terahertz frequencies is presented. In the design, antenna and mixer are combined into an entity, based on the technology in which millimeter-wave horn antenna arrays have been fabricated in silicon wafers. It consists of a set of forward- and backward-looking horns made with a set of silicon wafers. The front side is used to receive incoming signal, and the back side is used to feed local oscillator signal. Intermediate frequency is led out from the side of the array. Signal received by the horn array is picked up by antenna probes suspended on thin silicon-oxynitride membranes inside the horns. Mixer diodes will be located on the membranes inside the horns. Modeling of such an antenna-mixer-array design is done on a scaled model at microwave frequencies. The impedance matching, RF and LO isolation, and patterns of the array have been tested and analyzed.

GHz Yb:KYW oscillators in time-resolved spectroscopy

NASA Astrophysics Data System (ADS)

Li, Changxiu; Krauß, Nico; Schäfer, Gerhard; Ebner, Lukas; Kliebisch, Oliver; Schmidt, Johannes; Winnerl, Stephan; Hettich, Mike; Dekorsy, Thomas

2018-02-01

A high-speed asynchronous optical sampling system (ASOPS) based on Yb:KYW oscillators with 1-GHz repetition rate is reported. Two frequency-offset-stabilized diode-pumped Yb:KYW oscillators are employed as pump and probe source, respectively. The temporal resolution of this system within 1-ns time window is limited to 500 fs and the noise floor around 10-6 (ΔR/R) close to the shot-noise level is obtained within an acquisition time of a few seconds. Coherent acoustic phonons are investigated by measuring multilayer semiconductor structures with multiple quantum wells and aluminum/silicon membranes in this ASOPS system. A wavepacket-like phonon sequence at 360 GHz range is detected in the semiconductor structures and a decaying sequence of acoustic oscillations up to 200 GHz is obtained in the aluminum/silicon membranes. Coherent acoustic phonons generated from semiconductor structures are further manipulated by a double pump scheme through pump time delay control.

Influence of Lipid Membrane Rigidity on Properties of Supporting Polymer

PubMed Central

Jablin, Michael S.; Dubey, Manish; Zhernenkov, Mikhail; Toomey, Ryan; Majewski, Jarosław

2011-01-01

Temperature-sensitive hydrogel polymers are utilized as responsive layers in various applications. Although the polymer's native characteristics have been studied extensively, details concerning its properties during interaction with biorelated structures are lacking. This work investigates the interaction between a thermoresponsive polymer cushion and different lipid membrane capping layers probed by neutron reflectometry. N-isopropylacrylamide copolymerized with methacroylbenzophenone first supported a lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and subsequently 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The polymer-membrane systems were investigated above and below the polymer transition temperature (37 and 25°C). Although the same cushion supported each lipid membrane, the polymer hydration profile and thickness were markedly different for DPPE and DPPC systems. Because DPPE and DPPC have different bending rigidities, these results establish that the polymer-membrane interaction is critically mediated by the mechanics of the membrane, providing better insight into cell-hydrogel interactions. PMID:21723822

Studies on electronic structure of interfaces between Ag and gelatin for stabilization of Ag nanoparticles

NASA Astrophysics Data System (ADS)

Tani, Tadaaki; Uchida, Takayuki

2015-06-01

Extremely high stability of Ag nanoparticles in photographic materials has forced us to study the electronic structures of the interfaces between thin layers of Ag, Au, and Pt and their surface membranes in ambient atmosphere by photoelectron yield spectroscopy in air and Kelvin probe method. Owing to the Fermi level equalization between a metal layer and a membrane coming from air, the electron transfer took place from the membrane to Pt and Au layers and from an Ag layer to the membrane, giving the reason for poor stability of Ag nanoparticles in air. The control of the Fermi level of an Ag layer with respect to that of a gelatin membrane in air could be widely made according to Nernst’s equation by changing the pH and pAg values of an aqueous gelatin solution used to form the membrane, and thus available to stabilize Ag nanoparticles in a gelatin matrix.

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

PubMed Central

Andersen, O S; Feldberg, S; Nakadomari, H; Levy, S; McLaughlin, S

1978-01-01

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes. PMID:620077

Layer-by-layer cell membrane assembly

NASA Astrophysics Data System (ADS)

Matosevic, Sandro; Paegel, Brian M.

2013-11-01

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells.

PubMed

Zhao, Wenting; Hanson, Lindsey; Lou, Hsin-Ya; Akamatsu, Matthew; Chowdary, Praveen D; Santoro, Francesca; Marks, Jessica R; Grassart, Alexandre; Drubin, David G; Cui, Yi; Cui, Bianxiao

2017-08-01

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts 1

PubMed Central

Moeller, Curt H.; Mudd, J. Brian

1982-01-01

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:16662716

Flexural plate wave devices fabricated from silicon carbide membrane

NASA Astrophysics Data System (ADS)

Diagne, Ndeye Fama

Flexural Plate Wave (FPW) devices fabricated from Silicon Carbide (SiC) membranes are presented here which exhibit electrical and mechanical characteristics in its transfer functions that makes it very useful as a low voltage probe device capable of functioning in small areas that are commonly inaccessible to ordinary devices. The low input impedance characteristic of this current driven device makes it possible for it to operate at very low voltages, thereby reducing the hazards for flammable or explosive areas to be probed. The Flexural Plate Wave (FPW) devices are of a family of gravimetric type sensors that permit direct measurements of the mass of the vibrating element. The primary objective was to study the suitability of Silicon Carbide (SiC) membranes as a replacement of Silicon Nitride (SiN) membrane in flexural plate wave devices developed by Sandia National Laboratories. Fabrication of the Flexural Plate Wave devices involves the overlaying a silicon wafer with membranes of 3C-SiC thin film upon which conducting meander lines are placed. The input excitation energy is in the form of an input current. The lines of current along the direction of the conducting Meander Lines Transducer (MLTs) and the applied perpendicular external magnetic field set up a mechanical wave perpendicular to both, exciting the membrane by means of a Lorentz force, which in turn sets up flexural waves that propagate along the thin membrane. The physical dimensions, the mass density, the tension in the membrane and the meander spacing are physical characteristics that determine resonance frequency of the Flexural Plate Wave (FPW) device. Of primary interest is the determination of the resonant frequency of the silicon carbide membrane as functions of the device physical characteristic parameters. The appropriate transduction scheme with Meander Line Transducers (IDTs) are used to excite the membrane. Equivalent circuit models characterizing the reflection response S11 (amplitude and phase) for a one-port Flexural PlateWave device and the transmission response S21 of a two-port device are used for the development of the equivalent mechanical characteristics.

Indications for acceleration-dependent changes of membrane potential in the flagellate Euglena gracilis.

PubMed

Richter, P R; Schuster, M; Meyer, I; Lebert, M; Häder, D-P

2006-12-01

The effects of the calcium sequester EGTA on gravitactic orientation and membrane potential changes in the unicellular flagellate Euglena gracilis were investigated during a recent parabolic-flight experiment aboard of an Airbus A300. In the course of a flight parabola, an acceleration profile is achieved which yields subsequently about 20 s of hypergravity (1.8 g(n)), about 20 s of microgravity, and another 20 s of hypergravity phases. The movement behavior of the cells was investigated with real-time, computer-based image analysis. Membrane potential changes were detected with a newly developed photometer which measures absorption changes of the membrane potential-sensitive probe oxonol VI. To test whether the data obtained by the oxonol device were reliable, the signal of non-oxonol-labelled cells was recorded. In these samples, no absorption shift was detected. Changes of the oxonol VI signals indicate that the cells depolarize during acceleration (very obvious in the step from microgravity to hypergravity) and slightly hyperpolarize in microgravity, which can possibly be explained with the action of Ca-ATPases. These signals (mainly the depolarization) were significantly suppressed in the presence of EGTA (5 mM). Gravitaxis in parallel was also inhibited after addition of EGTA. Initially, negative gravitaxis was inverted into a positive one. Later, gravitaxis was almost undetectable.

Using Pre-Assessment and In-Class Questions to Change Student Understanding of Molecular Movements †

PubMed Central

Shi, J.; Knight, Jennifer K.; Chun, Hyonho; Guild, Nancy A.; Martin, Jennifer M.

2017-01-01

Understanding how different types of molecules move through cell membranes is a fundamental part of cell biology. To identify and address student misconceptions surrounding molecular movement through cell membranes, we surveyed student understanding on this topic using pre-class questions, in-class clicker questions, and subsequent exam questions in a large introductory biology course. Common misconceptions identified in student responses to the pre-class assessment questions were used to generate distractors for clicker questions. Two-tier diagnostic clicker questions were used to probe incoming common student misconceptions (first tier) and their reasoning (second tier). Two subsequent lectures with assessment clicker questions were used to help students construct a new framework to understand molecular movement through cell membranes. Comparison of pre-assessment and post-assessment (exam) performance showed dramatic improvement in students’ understanding of molecular movement: student answers to exam questions were 74.6% correct with correct reasoning while only 1.3% of the student answers were correct with correct reasoning on the pre-class assessment. Our results show that students’ conceptual understanding of molecular movement through cell membranes progressively increases through discussions of a series of clicker questions and suggest that this clicker-based teaching strategy was highly effective in correcting common student misconceptions on this topic. PMID:28512521

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

PubMed Central

Murphy, Sean C.; Fernandez-Pol, Sebastian; Chung, Paul H.; Prasanna Murthy, S. N.; Milne, Stephen B.; Salomao, Marcela; Brown, H. Alex; Lomasney, Jon W.; Mohandas, Narla

2007-01-01

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non–receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP2) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP2, although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection. PMID:17526861

Derivation of a closed form analytical expression for fluorescence recovery after photo bleaching in the case of continuous bleaching during read out

NASA Astrophysics Data System (ADS)

Endress, E.; Weigelt, S.; Reents, G.; Bayerl, T. M.

2005-01-01

Measurements of very slow diffusive processes in membranes, like the diffusion of integral membrane proteins, by fluorescence recovery after photo bleaching (FRAP) are hampered by bleaching of the probe during the read out of the fluorescence recovery. In the limit of long observation time (very slow diffusion as in the case of large membrane proteins), this bleaching may cause errors to the recovery function and thus provides error-prone diffusion coefficients. In this work we present a new approach to a two-dimensional closed form analytical solution of the reaction-diffusion equation, based on the addition of a dissipative term to the conventional diffusion equation. The calculation was done assuming (i) a Gaussian laser beam profile for bleaching the spot and (ii) that the fluorescence intensity profile emerging from the spot can be approximated by a two-dimensional Gaussian. The detection scheme derived from the analytical solution allows for diffusion measurements without the constraint of observation bleaching. Recovery curves of experimental FRAP data obtained under non-negligible read-out bleaching for native membranes (rabbit endoplasmic reticulum) on a planar solid support showed excellent agreement with the analytical solution and allowed the calculation of the lipid diffusion coefficient.

The early days of blotting.

PubMed

Southern, Edwin

2015-01-01

The history of the development of DNA blotting is described in this chapter. DNA blotting, involving the transfer of electrophoretically separated DNA fragments to a membrane support through capillary action, is also known as Southern blotting. This procedure enables the detection of a specific DNA sequence by hybridization with probes. The term Southern blotting led to a "geographic" naming tradition, with RNA blotting bearing the name Northern blotting and protein transfer to membranes becoming known as Western blotting.

One-dimensional surface-plasmon gratings for the excitation of intersubband polaritons in suspended membranes

NASA Astrophysics Data System (ADS)

Degl'Innocenti, R.; Zanotto, S.; Tredicucci, A.; Biasiol, G.; Sorba, L.

2011-12-01

We present the observation of the strong light-matter coupling regime between intersubband transitions of semiconductor quantum wells and the plasmonic-like resonances of a one dimensional metallic grating. Polariton spectra have been recorded in transmission employing a suspended membrane sample and are consistent with theoretical calculations. This arrangement, avoiding the complexity of dispersive substrate, is particularly attractive for the development of time-resolved pump-probe experiments.

A Novel Cassette Method for Probe Evaluation in the Designed Biochips

PubMed Central

Zinkevich, Vitaly; Sapojnikova, Nelly; Mitchell, Julian; Kartvelishvili, Tamar; Asatiani, Nino; Alkhalil, Samia; Bogdarina, Irina; Al-Humam, Abdulmohsen A.

2014-01-01

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip. PMID:24897111

Biosensors for spatiotemporal detection of reactive oxygen species in cells and tissues.

PubMed

Erard, Marie; Dupré-Crochet, Sophie; Nüße, Oliver

2018-05-01

Redox biology has become a major issue in numerous areas of physiology. Reactive oxygen species (ROS) have a broad range of roles from signal transduction to growth control and cell death. To understand the nature of these roles, accurate measurement of the reactive compounds is required. An increasing number of tools for ROS detection is available; however, the specificity and sensitivity of these tools are often insufficient. Furthermore, their specificity has been rarely evaluated in complex physiological conditions. Many ROS probes are sensitive to environmental conditions in particular pH, which may interfere with ROS detection and cause misleading results. Accurate detection of ROS in physiology and pathophysiology faces additional challenges concerning the precise localization of the ROS and the timing of their production and disappearance. Certain ROS are membrane permeable, and certain ROS probes move across cells and organelles. Targetable ROS probes such as fluorescent protein-based biosensors are required for accurate localization. Here we analyze these challenges in more detail, provide indications on the strength and weakness of current tools for ROS detection, and point out developments that will provide improved ROS detection methods in the future. There is no universal method that fits all situations in physiology and cell biology. A detailed knowledge of the ROS probes is required to choose the appropriate method for a given biological problem. The knowledge of the shortcomings of these probes should also guide the development of new sensors.

Ultra-small particles of iron oxide as peroxidase for immunohistochemical detection

NASA Astrophysics Data System (ADS)

Wu, Yihang; Song, Mengjie; Xin, Zhuang; Zhang, Xiaoqing; Zhang, Yu; Wang, Chunyu; Li, Suyi; Gu, Ning

2011-06-01

Dimercaptosuccinic acid (DMSA) modified ultra-small particles of iron oxide (USPIO) were synthesized through a two-step process. The first step: oleic acid (OA) capped Fe3O4 (OA-USPIO) were synthesized by a novel oxidation coprecipitation method in H2O/DMSO mixing system, where DMSO acts as an oxidant simultaneously. The second step: OA was replaced by DMSA to obtain water-soluble nanoparticles. The as-synthesized nanoparticles were characterized by TEM, FTIR, TGA, VSM, DLS, EDS and UV-vis. Hydrodynamic sizes and Peroxidase-like catalytic activity of the nanoparticles were investigated. The hydrodynamic sizes of the nanoparticles (around 24.4 nm) were well suited to developing stable nanoprobes for bio-detection. The kinetic studies were performed to quantitatively evaluate the catalytic ability of the peroxidase-like nanoparticles. The calculated kinetic parameters indicated that the DMSA-USPIO possesses high catalytic activity. Based on the high activity, immunohistochemical experiments were established: using low-cost nanoparticles as the enzyme instead of expensive HRP, Nimotuzumab was conjugated onto the surface of the nanoparticles to construct a kind of ultra-small nanoprobe which was employed to detect epidermal growth factor receptor (EGFR) over-expressed on the membrane of esophageal cancer cell. The proper sizes of the probes and the result of membranous immunohistochemical staining suggest that the probes can be served as a useful diagnostic reagent for bio-detection.

Comparative evaluation of transmembrane ion transport due to monopolar and bipolar nanosecond, high-intensity electroporation pulses based on full three-dimensional analyses

NASA Astrophysics Data System (ADS)

Hu, Q.; Joshi, R. P.

2017-07-01

Electric pulse driven membrane poration finds applications in the fields of biomedical engineering and drug/gene delivery. Here we focus on nanosecond, high-intensity electroporation and probe the role of pulse shape (e.g., monopolar-vs-bipolar), multiple electrode scenarios, and serial-versus-simultaneous pulsing, based on a three-dimensional time-dependent continuum model in a systematic fashion. Our results indicate that monopolar pulsing always leads to higher and stronger cellular uptake. This prediction is in agreement with experimental reports and observations. It is also demonstrated that multi-pronged electrode configurations influence and increase the degree of cellular uptake.

Plasmonic Techniques for Viral Membrane Characterization

NASA Astrophysics Data System (ADS)

Feizpour, Amin

The lipid bilayer membrane of enveloped viruses, such as human immunodeficiency virus type 1 (HIV-1), plays an important role in key steps of the infection, including cell binding and uptake. Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. In this dissertation, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different virus-like particles (VLPs) using small sample sizes is introduced. The assay evaluates both scattering intensity and resonance wavelength and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. The performed studies reveal significant differences in the membrane of HIV-1 and Ebola VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers. In addition, this technique was used in another application to improve the understanding of the relationship between the membrane PS lipid and the infectivity of HIV-2 and murine leukemia virus (MLV). The composition of the membrane, in particular the cholesterol (chol) content, determines its fluidity. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of probing the membrane fluidity on the single-virus level are required. In this dissertation, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed a plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated, for the first time, the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature on the single-VLP level. Chol extraction studies with different methyl-beta-cyclodextrin (MbetaCD) concentrations yielded a gradual decrease in polarization fluctuations as function of time. The PFTM revealed chol content and fluidity heterogeneities of an HIV-1 VLP population.

Membrane Composition Tunes the Outer Hair Cell Motor

NASA Astrophysics Data System (ADS)

Rajagopalan, L.; Sfondouris, J.; Oghalai, J. S.; Pereira, F. A.; Brownell, W. E.

2009-02-01

Cholesterol and docosahexaenoic acid (DHA), an ω-3 fatty acid, affect membrane mechanical properties in different ways and modulate the function of membrane proteins. We have probed the functional consequence of altering cholesterol and DHA levels in the membranes of OHCs and prestin expressing HEK cells. Large, dynamic and reversible changes in prestin-associated charge movement and OHC motor activity result from altering the concentration of membrane cholesterol. Increasing membrane cholesterol shifts the q/V function ~ 50 mV in the hyperpolarizing direction, possibly a response related to increases in membrane stiffness. The voltage shift is linearly related to total membrane cholesterol. Increasing cholesterol also decreases the total charge moved in a linear fashion. Decreasing membrane cholesterol shifts the q/V function ~ 50 mV in the depolarizing direction with little or no effect on the amount of charge moved. In vivo increases in membrane cholesterol transiently increase but ultimately lead to decreases in DPOAE. Docosahexaenoic acid shifts the q/V function in the hyperpolarizing direction < 15 mV and increases total charge moved. Tuning of cochlear function by membrane cholesterol contributes to the exquisite temporal and frequency processing of mammalian hearing by optimizing the cochlear amplifier.

Apparent Softening of Wet Graphene Membranes on a Microfluidic Platform.

PubMed

Ferrari, Gustavo A; de Oliveira, Alan B; Silvestre, Ive; Matos, Matheus J S; Batista, Ronaldo J C; Fernandes, Thales F D; Meireles, Leonel M; Eliel, Gomes S N; Chacham, Helio; Neves, Bernardo R A; Lacerda, Rodrigo G

2018-05-22

Graphene is regarded as the toughest two-dimensional material (highest in-plane elastic properties) and, as a consequence, it has been employed/proposed as an ultrathin membrane in a myriad of microfluidic devices. Yet, an experimental investigation of eventual variations on the apparent elastic properties of a suspended graphene membrane in contact with air or water is still missing. In this work, the mechanical response of suspended monolayer graphene membranes on a microfluidic platform is investigated via scanning probe microscopy experiments. A high elastic modulus is measured for the membrane when the platform is filled with air, as expected. However, a significant apparent softening of graphene is observed when water fills the microfluidic system. Through molecular dynamics simulations and a phenomenological model, we associate such softening to a water-induced uncrumpling process of the suspended graphene membrane. This result may bring substantial modifications on the design and operation of microfluidic devices which exploit pressure application on graphene membranes.

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

PubMed

Malabed, Raymond; Hanashima, Shinya; Murata, Michio; Sakurai, Kaori

2017-12-01

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Temperature effects on nanostructure and mechanical properties of single-nanoparticle thick membranes.

DOE PAGES

Salerno, Kenneth Michael; Grest, Gary S.

2015-04-30

In this study, the properties of mechanically stable single-nanoparticle (NP)-thick membranes have largely been studied at room temperature. How these membranes soften as nanoparticle ligands disorder with increasing temperature is unknown. Molecular dynamics simulations are used to probe the temperature dependence of the mechanical and nanostructural properties of nanoparticle membranes made of 6 nm diameter Au nanoparticles coated with dodecanethiol ligands and terminated with either methyl (CH 3) or carboxyl (COOH) terminal groups. For methyl-terminated ligands, interactions along the alkane chain provide mechanical stiffness, with a Young's modulus of 1.7 GPa at 300 K. For carboxyl-terminated chains, end-group interactions aremore » significant, producing stiffer membranes at all temperatures, with a Young's modulus of 3.8 GPa at 300 K. For both end-group types, membrane stiffness is reduced to zero at about 400 K. Ligand structure and mechanical properties of membranes at 300 K that have been annealed at 400 K are comparable to samples that do not undergo thermal annealing.« less

FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells.

PubMed

Nicovich, Philip R; Kwiatek, Joanna M; Ma, Yuanqing; Benda, Aleš; Gaus, Katharina

2018-06-19

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

PubMed

Maraviglia, B; Herring, F G; Weeks, G; Godin, D V

1979-01-01

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.

Tensiometer methods

DOEpatents

Grover, Blair K.; Hubbell, Joel M.; Sisson, James B.; Casper, William L.

2005-12-20

A method for collecting data regarding a matric potential of a media includes providing a tensiometer having a stainless steel tensiometer casing, the stainless steel tensiometer casing comprising a tip portion which includes a wetted porous stainless steel membrane through which a matric potential of a media is sensed; driving the tensiometer into the media using an insertion tube comprising a plurality of probe casing which are selectively coupled to form the insertion tube as the tensiometer is progressively driven deeper into the media, wherein the wetted porous stainless steel membrane is in contact with the media; and sensing the matric potential the media exerts on the wetted porous stainless steel membrane by a pressure sensor in fluid hydraulic connection with the porous stainless steel membrane. A tensiometer includes a stainless steel casing.

Dynamics of a Room Temperature Ionic Liquid in Supported Ionic Liquid Membranes vs the Bulk Liquid: 2D IR and Polarized IR Pump-Probe Experiments.

PubMed

Shin, Jae Yoon; Yamada, Steven A; Fayer, Michael D

2017-01-11

Supported ionic liquid membranes (SILMs) are membranes that have ionic liquids impregnated in their pores. SILMs have been proposed for advanced carbon capture materials. Two-dimensional infrared (2D IR) and polarization selective IR pump-probe (PSPP) techniques were used to investigate the dynamics of reorientation and spectral diffusion of the linear triatomic anion, SeCN - , in poly(ether sulfone) (PES) membranes and room-temperature ionic liquid (RTIL), 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EmimNTf 2 ). The dynamics in the bulk EmimNTf 2 were compared to its dynamics in the SILM samples. Two PES membranes, PES200 and PES30, have pores with average sizes, ∼300 nm and ∼100 nm, respectively. Despite the relatively large pore sizes, the measurements reveal that the reorientation of SeCN - and the RTIL structural fluctuations are substantially slower in the SILMs than in the bulk liquid. The complete orientational randomization, slows from 136 ps in the bulk to 513 ps in the PES30. 2D IR measurements yield three time scales for structural spectral diffusion (SSD), that is, the time evolution of the liquid structure. The slowest decay constant increases from 140 ps in the bulk to 504 ps in the PES200 and increases further to 1660 ps in the PES30. The results suggest that changes at the interface propagate out and influence the RTIL structural dynamics even more than a hundred nanometers from the polymer surface. The differences between the IL dynamics in the bulk and in the membranes suggest that studies of bulk RTIL properties may be poor guides to their use in SILMs in carbon capture applications.

Molecular dynamics simulations of membrane proteins and their interactions: from nanoscale to mesoscale.

PubMed

Chavent, Matthieu; Duncan, Anna L; Sansom, Mark Sp

2016-10-01

Molecular dynamics simulations provide a computational tool to probe membrane proteins and systems at length scales ranging from nanometers to close to a micrometer, and on microsecond timescales. All atom and coarse-grained simulations may be used to explore in detail the interactions of membrane proteins and specific lipids, yielding predictions of lipid binding sites in good agreement with available structural data. Building on the success of protein-lipid interaction simulations, larger scale simulations reveal crowding and clustering of proteins, resulting in slow and anomalous diffusional dynamics, within realistic models of cell membranes. Current methods allow near atomic resolution simulations of small membrane organelles, and of enveloped viruses to be performed, revealing key aspects of their structure and functionally important dynamics. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

PubMed Central

Muangchuen, Ajima; Chaumpluk, Piyasak; Suriyasomboon, Annop; Ekgasit, Sanong

2014-01-01

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease. PMID:25111239

Colorimetric detection of Ehrlichia canis via nucleic acid hybridization in gold nano-colloids.

PubMed

Muangchuen, Ajima; Chaumpluk, Piyasak; Suriyasomboon, Annop; Ekgasit, Sanong

2014-08-08

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease.

Growth Suppression and Therapy Sensitization of Breast Cancer

DTIC Science & Technology

1999-07-01

northern analysis, the same membrane was hybridized successively with JNKI probe, JNK2 probe and the G3PDH probe. Figure 10. A549 cells were treated with the...90 0 00 z SO E 40 04. z 20 to JNK2 Oligonucleotide n* 47 JNK1AS TREATED 2 JNlK2AS TREATED. 0) c a 0 mRNA mRNA JNK1 J&,I ~R JNK1 JNK2 JNK2 G3PDH SI... G3PDH 48 iil vý 0 z z Z O! zz C13~ A.LOR 496 Reference #28 PREFERENTIAL PLATINATION OF AN ACTIVATED CELLULAR PROMOTER BY CIS-DIAMMINEDICHLOROPLATINUM

Development of an optical Zn 2+ probe based on a single fluorescent protein

DOE PAGES

Qin, Yan; Sammond, Deanne W.; Braselmann, Esther; ...

2016-07-28

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn 2+. Here we present Green Zinc Probe (GZnP), a novel genetically encoded Zn 2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created tomore » include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1 which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn 2+ in HeLa cells (F max/F min = 2.6, K d = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn 2+ (F max/F min = 2.2). In conclusion, this sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn 2+ function.« less

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions.

PubMed

Bashford, C L; Morgan, C G; Radda, G K

1976-03-05

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystalline-liquid crystalline phase transitions in phospholipid vesicles of dimyristoly and dipalmitoly phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidylcholine show lateral phase separation and in this system the probes parition preferentially into the more 'fluid' phase. In systems that have been reported to contain 'short range order' or 'liquid clustering', such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.

Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

PubMed Central

Narihiro, Takashi; Sekiguchi, Yuji

2011-01-01

Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight

NASA Technical Reports Server (NTRS)

Weinstein, S.; Pane, D.; Warren-Rhodes, K.; Cockell, C.; Ernst, L. A.; Minkley, E.; Fisher, G.; Emani, S.; Wettergreen, D. S.; Wagner, M.

2005-01-01

We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

System dynamics of subcellular transport.

PubMed

Chen, Vivien Y; Khersonsky, Sonya M; Shedden, Kerby; Chang, Young Tae; Rosania, Gus R

2004-01-01

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

Acceleration of Lateral Equilibration in Mixed Lipid Bilayers Using Replica Exchange with Solute Tempering

PubMed Central

2015-01-01

The lateral heterogeneity of cellular membranes plays an important role in many biological functions such as signaling and regulating membrane proteins. This heterogeneity can result from preferential interactions between membrane components or interactions with membrane proteins. One major difficulty in molecular dynamics simulations aimed at studying the membrane heterogeneity is that lipids diffuse slowly and collectively in bilayers, and therefore, it is difficult to reach equilibrium in lateral organization in bilayer mixtures. Here, we propose the use of the replica exchange with solute tempering (REST) approach to accelerate lateral relaxation in heterogeneous bilayers. REST is based on the replica exchange method but tempers only the solute, leaving the temperature of the solvent fixed. Since the number of replicas in REST scales approximately only with the degrees of freedom in the solute, REST enables us to enhance the configuration sampling of lipid bilayers with fewer replicas, in comparison with the temperature replica exchange molecular dynamics simulation (T-REMD) where the number of replicas scales with the degrees of freedom of the entire system. We apply the REST method to a cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer mixture and find that the lateral distribution functions of all molecular pair types converge much faster than in the standard MD simulation. The relative diffusion rate between molecules in REST is, on average, an order of magnitude faster than in the standard MD simulation. Although REST was initially proposed to study protein folding and its efficiency in protein folding is still under debate, we find a unique application of REST to accelerate lateral equilibration in mixed lipid membranes and suggest a promising way to probe membrane lateral heterogeneity through molecular dynamics simulation. PMID:25328493

Measurement of the membrane dipole electric field in DMPC vesicles using vibrational shifts of p-cyanophenylalanine and molecular dynamics simulations.

PubMed

Shrestha, Rebika; Cardenas, Alfredo E; Elber, Ron; Webb, Lauren J

2015-02-19

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the headgroup region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction of the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped toward the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ∼3 cm(-1) greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in an absolute magnitude of 8-11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed.

Measurement of the Membrane Dipole Electric Field in DMPC Vesicles Using Vibrational Shifts of p-Cyanophenylalanine and Molecular Dynamics Simulations

PubMed Central

Shrestha, Rebika; Cardenas, Alfredo E.; Elber, Ron; Webb, Lauren J.

2015-01-01

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped towards the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ~3 cm−1 greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in a value of 8 – 11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed. PMID:25602635

Acceleration of Lateral Equilibration in Mixed Lipid Bilayers Using Replica Exchange with Solute Tempering.

PubMed

Huang, Kun; García, Angel E

2014-10-14

The lateral heterogeneity of cellular membranes plays an important role in many biological functions such as signaling and regulating membrane proteins. This heterogeneity can result from preferential interactions between membrane components or interactions with membrane proteins. One major difficulty in molecular dynamics simulations aimed at studying the membrane heterogeneity is that lipids diffuse slowly and collectively in bilayers, and therefore, it is difficult to reach equilibrium in lateral organization in bilayer mixtures. Here, we propose the use of the replica exchange with solute tempering (REST) approach to accelerate lateral relaxation in heterogeneous bilayers. REST is based on the replica exchange method but tempers only the solute, leaving the temperature of the solvent fixed. Since the number of replicas in REST scales approximately only with the degrees of freedom in the solute, REST enables us to enhance the configuration sampling of lipid bilayers with fewer replicas, in comparison with the temperature replica exchange molecular dynamics simulation (T-REMD) where the number of replicas scales with the degrees of freedom of the entire system. We apply the REST method to a cholesterol and 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) bilayer mixture and find that the lateral distribution functions of all molecular pair types converge much faster than in the standard MD simulation. The relative diffusion rate between molecules in REST is, on average, an order of magnitude faster than in the standard MD simulation. Although REST was initially proposed to study protein folding and its efficiency in protein folding is still under debate, we find a unique application of REST to accelerate lateral equilibration in mixed lipid membranes and suggest a promising way to probe membrane lateral heterogeneity through molecular dynamics simulation.

Modular 3D-Printed Soil Gas Probes

NASA Astrophysics Data System (ADS)

Good, S. P.; Selker, J. S.; Al-Qqaili, F.; Lopez, M.; Kahel, L.

2016-12-01

ABSTRACT: Extraction of soil gas is required for a variety of applications in earth sciences and environmental engineering. However, commercially available probes can be costly and are typically limited to a single depth. Here, we present the open-source design and lab testing of a soil gas probe with modular capabilities that allow for the vertical stacking of gas extraction points at different depths in the soil column. The probe modules consist of a 3D printed spacer unit and hydrophobic gas permeable membrane made of high density Polyethylene with pore sizes 20-40 microns. Each of the modular spacer units contain both a gas extraction line and gas input line for the dilution of soil gases if needed. These 2-inch diameter probes can be installed in the field quickly with a hand auger and returned to at any frequency to extract soil gas from desired soil depths. The probes are tested through extraction of soil pore water vapors with distinct stable isotope ratios.

An Electrochemical DNA Microbiosensor Based on Succinimide-Modified Acrylic Microspheres

PubMed Central

Ulianas, Alizar; Heng, Lee Yook; Hanifah, Sharina Abu; Ling, Tan Ling

2012-01-01

An electrochemical microbiosensor for DNA has been fabricated based on new acrylic microspheres modified with reactive N-acryloxysuccinimide (NAS) functional groups. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized in an emulsion form with a simple one-step photopolymerization technique. Aminated DNA probe was attached to the succinimde functional group of the acrylic microspheres via covalent bonding. The hybridization of the immobilized DNA probe with the complementary DNA was studied by differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, pH, type of ions, buffer concentrations, ionic strength, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a linear response range to target DNA over a wide concentration range of 1.0 × 10−16 and 1.0 × 10−8 M with a lower limit of detection (LOD) of 9.46 × 10−17 M (R2 = 0.97). This DNA microbiosensor showed good reproducibility with 2.84% RSD (relative standard deviation) (n = 3). Application of the NAS-modified acrylic microspheres in the construction of DNA microbiosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices. PMID:22778594

Probing the temperature-dependent changes of the interfacial hydration and viscosity of Tween20 : cholesterol (1 : 1) niosome membrane using fisetin as a fluorescent molecular probe.

PubMed

Mishra, Jhili; Swain, Jitendriya; Mishra, Ashok Kumar

2018-05-16

A detailed photophysical study of fisetin in a Tween20 : cholesterol (1 : 1) niosome membrane has been carried out. Fisetin is found to partition well into the Tween20 : cholesterol (1 : 1) niosome membrane at low temperature (Kp = 2.7 × 104 M-1 at 10 °C). Cetylpyridinium chloride quenching study confirms the location of fisetin molecules in the interfacial domain of Tween20 : cholesterol (1 : 1) niosome membrane. The emission from the prototropic forms of fisetin (neutral form, excited state anion, ground state anion and phototautomer form) is found to sensitively reflect the local heterogeneities in Tween20 : cholesterol (1 : 1) niosome membrane. The shift in anionic emission maximum with variation in temperature shows the sensitivity of fisetin towards water accessibility at the interfacial domain of Tween20 : cholesterol (1 : 1) niosome membrane. Zeta potential value confirms that there is no role of surface charge in the multiple prototropism of fisetin in Tween20 : cholesterol (1 : 1) niosome membrane. The microviscosity changes with temperature, as reflected in fluorescence anisotropy values of fisetin phototautomeric species FT*, give information about the temperature-induced changes in the motional resistance offered by the interfacial domain of the niosomal membrane to small molecules. A temperature-dependent fluorescence lifetime study confirms the distribution of FT* in the two different sites of niosomal interfacial domain, i.e. water-deficient inner site and water-accessible outer site. This heterogeneity in distribution of FT* is further confirmed through time-resolved fluorescence anisotropy decay resulting in two different rotational time constants (faster component of ∼1.04 ns originates from water-accessible outer site and slower component of ∼16.50 ns originates from water-deficient inner site). The interfacial location of fisetin in Tween20 : cholesterol (1 : 1) niosome membrane has an important implication with regards to antioxidant activity as confirmed from a DPPH radical scavenging study.

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2007-02-01

saponin (Calbiochem, San Diego, CA) in PBS. Results, Significance, Obstacles and Alternative Approaches: We have generated several different fluorescent...1 integrin antibody P4C10 (Life technologies ). We will conjugate the fluorescent probes to these functional blocking antibodies for live cell...characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids. Int. J. Cancer. 62:552-558. 1995. 9. Wright GL Jr

Calcium and Mitosis

NASA Technical Reports Server (NTRS)

Hepler, P.

1983-01-01

Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane*

PubMed Central

Ji, Chen; Zhang, Yongdeng; Xu, Pingyong; Xu, Tao; Lou, Xuelin

2015-01-01

Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2-enriched membrane patches/domains of diverse sizes (383 ± 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) showed similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale. PMID:26396197

Interactions of beta-blockers with model lipid membranes: molecular view of the interaction of acebutolol, oxprenolol, and propranolol with phosphatidylcholine vesicles by time-dependent fluorescence shift and molecular dynamics simulations.

PubMed

Först, Gesche; Cwiklik, Lukasz; Jurkiewicz, Piotr; Schubert, Rolf; Hof, Martin

2014-08-01

Since pharmacokinetic and pharmacodynamic activities of drugs are often related to their interactions with biomembranes, it is of high interest to establish an approach for the characterization of these interactions at the molecular level. For the present study, beta-blockers (oxprenolol, propranolol, and acebutolol) were selected due to their well described nonspecific membrane effects (NME). Their interactions with model lipid membranes composed of palmitoyloleoylphosphatidylcholine (POPC) were studied using Time-Dependent Fluorescence Shift (TDFS) and Generalized Polarization (GP) as well as molecular dynamics (MD) simulations. Liposomal vesicles were labeled with fluorescent membrane polarity probes (Laurdan, Prodan, and Dtmac). Increasing beta-blocker concentrations (0-10 mM for acebutolol and oxprenolol, and 0-1.5 mM for propranolol) significantly rigidifies the lipid bilayer at the glycerol and headgroup level, which was detected in the steady-state and in the time-resolved fluorescence data. The effects of propranolol were considerably stronger than those of the two other beta-blockers. The addition of fluorescent probes precisely located at different levels within the lipid bilayer revealed the insertion of the beta-blockers into the POPC bilayer at the glycerol backbone level, which was further confirmed by MD simulations in the case of propranolol. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential natural organic matter fouling of ceramic versus polymeric ultrafiltration membranes.

PubMed

Lee, Seung-Jin; Kim, Jae-Hong

2014-01-01

Ceramic ultrafiltration membranes has drawn increasing attention in drinking water treatment sectors as an alternative to traditional polymeric counterparts, yet only limited information has been made available about the characteristics of ceramic membrane fouling by natural organic matter. The effects of solution chemistry including ionic strength, divalent ion concentration and pH on the flux behavior were comparatively evaluated for ceramic and polymeric ultrafiltration of synthetic water containing model natural organic matter. Filtration characteristics were further probed via resistance-in-series model analysis, fouling visualization using quantum dots, batch adsorption test, contact angle measurement, solute-membrane surface adhesion force measurement, and quantitative comparison of fouling characteristics between ceramic and polymeric membranes. The results collectively suggested that the effects of solution chemistry on fouling behavior of ceramic membranes were generally similar to polymeric counterparts in terms of trends, while the extent varied significantly depending on water quality parameters. Lower fouling tendency and enhanced cleaning efficiency were observed with the ceramic membrane, further promoting the potential for ceramic membrane application to surface water treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Multiplexed measurement of protein diffusion in Caenorhabditis elegans embryos with SPIM-FCS

NASA Astrophysics Data System (ADS)

Struntz, Philipp; Weiss, Matthias

2016-02-01

Quantifying the diffusion behavior of proteins in different environments, e.g. on cellular membranes, is a key step in uncovering the vital action of protein networks in living organisms. While several established techniques for local diffusion measurements exist, the life sciences are currently in need of a multiplexed, i.e. spatially parallelized, data acquisition that allows for obtaining diffusion maps with high spatiotemporal resolution. Following this demand, the combination of camera-based single-plane illumination microscopy (SPIM) and fluorescence correlation spectroscopy (FCS) has recently emerged as a promising approach. So far, SPIM-FCS has mainly been used to assess the diffusion of soluble particles and proteins in vitro and in culture cells, but due to a particularly low photobleaching and -toxicity the method is also well applicable to developmental organisms. Here, we have probed the performance of SPIM-FCS on an established developmental model organism, the small nematode Caenorhabditis elegans. In particular, we have quantified the diffusion of the peripheral membrane protein PLC1δ 1 in the embryo’s cytoplasm and on the plasma membrane. As a result, we were able to derive diffusion maps of PLC1δ 1 in both compartments in multiple individuals, showing the spatially varying diffusion coefficients across the embryo. Our data also report on the dissociation kinetics of PLC1δ 1 from the plasma membrane, hence underlining that SPIM-FCS can be used to explore key features of peripheral membrane proteins in fragile developmental model organisms.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

NASA Astrophysics Data System (ADS)

Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten

2016-09-01

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Determination of membrane disruption and genomic DNA binding of cinnamaldehyde to Escherichia coli by use of microbiological and spectroscopic techniques.

PubMed

He, Tian-Fu; Zhang, Zhi-Hong; Zeng, Xin-An; Wang, Lang-Hong; Brennan, Charles S

2018-01-01

This work was aimed to investigate the antibacterial action of cinnamaldehyde (CIN) against Escherichia coli ATCC 8735 (E. coli) based on membrane fatty acid composition analysis, alterations of permeability and cell morphology as well as interaction with genomic DNA. Analysis of membrane fatty acids using gas chromatography-mass spectrometry (GC-MS) revealed that the proportion of unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were the major fatty acids in plasmic membrane, and their levels were significantly changed after exposure of E. coli to CIN at low concentrations. For example, the proportion of UFA decreased from 39.97% to 20.98%, while the relative content of SFA increased from 50.14% to 67.80% as E. coli was grown in increasing concentrations of CIN (from 0 to 0.88mM). Scanning electron microscopy (SEM) showed that the morphology of E. coli cells to be wrinkled, distorted and even lysed after exposure to CIN, which therefore decreased the cell viability. The binding of CIN to genomic DNA was probed using fluorescence, UV-Visible absorption spectra, circular dichroism, molecular modeling and atomic force microscopy (AFM). Results indicated that CIN likely bound to the minor groove of genomic DNA, and changed the secondary structure and morphology of this biomacromolecule. Therefore, CIN can be deem as a kind of natural antimicrobial agents, which influence both cell membrane and genomic DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Submillisecond Dynamics of Mastoparan X Insertion into Lipid Membranes.

PubMed

Schuler, Erin E; Nagarajan, Sureshbabu; Dyer, R Brian

2016-09-01

The mechanism of protein insertion into a lipid bilayer is poorly understood because the kinetics of this process is difficult to measure. We developed a new approach to study insertion of the antimicrobial peptide Mastoparan X into zwitterionic lipid vesicles, using a laser-induced temperature-jump to initiate insertion on the microsecond time scale and infrared and fluorescence spectroscopies to follow the kinetics. Infrared probes the desolvation of the peptide backbone and yields biphasic kinetics with relaxation lifetimes of 12 and 117 μs, whereas fluorescence probes the intrinsic tryptophan residue located near the N-terminus and yields a single exponential phase with a lifetime of 440 μs. Arrhenius analysis of the temperature-dependent rates yields an activation energy for insertion of 96 kJ/mol. These results demonstrate the complexity of the insertion process and provide mechanistic insight into the interplay between peptides and the lipid bilayer required for peptide transport across cellular membranes.

The metabolism of the phosphonium analogue of choline in cultured cells. A useful nuclear-magnetic-resonance probe for membrane phosphatidylcholine.

PubMed Central

Sim, E; Pasternak, C A

1976-01-01

1. Replacement of choline by the phosphonium analogue does not affect the growth rate of P815Y, NIL, 3T3, and SV40/3T3 cells in culture. 2. The fatty acid composition of the resulting phosphonium phosphatidylcholine is similar to that of phosphatidylcholine. 3. The rate of synthesis and degradation of phosphatidylcholine and of the phosphonium analogue are similar. 4. Phospholipid-exchange protein does not distinguish between phosphatidylcholine and the phosphonium analogue. 5. By contrast, incorporation of phosphonium choline into sphingomyelin occurs to only a minor extent. 6. It is concluded that, since the enzymes involved in the turnover of phosphatidylcholine do not discriminate between quaternary N and quaternary P in the polar head-group region, phosphonium choline should prove to be a useful probe for 31P nuclear-magnetic-resonance (n.m.r.) studies of natural membranes. PMID:1275902

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

PubMed

Dufrêne, Y F

2001-02-01

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

Horseradish Peroxidase-Encapsulated Hollow Silica Nanospheres for Intracellular Sensing of Reactive Oxygen Species

NASA Astrophysics Data System (ADS)

Chen, Hsin-Yi; Wu, Si-Han; Chen, Chien-Tsu; Chen, Yi-Ping; Chang, Feng-Peng; Chien, Fan-Ching; Mou, Chung-Yuan

2018-04-01

Reactive oxygen species (ROS) have crucial roles in cell signaling and homeostasis. Overproduction of ROS can induce oxidative damage to various biomolecules and cellular structures. Therefore, developing an approach capable of monitoring and quantifying ROS in living cells is significant for physiology and clinical diagnoses. Some cell-permeable fluorogenic probes developed are useful for the detection of ROS while in conjunction with horseradish peroxidase (HRP). Their intracellular scenario is however hindered by the membrane-impermeable property of enzymes. Herein, a new approach for intracellular sensing of ROS by using horseradish peroxidase-encapsulated hollow silica nanospheres (designated HRP@HSNs), with satisfactory catalytic activity, cell membrane permeability, and biocompatibility, was prepared via a microemulsion method. These HRP@HSNs, combined with selective probes or targeting ligands, could be foreseen as ROS-detecting tools in specific organelles or cell types. As such, dihydrorhodamine 123-coupled HRP@HSNs were used for the qualitative and semi-quantitative analysis of physiological H2O2 levels in activated RAW 264.7 macrophages. We envision that this HSNs encapsulating active enzymes can be conjugated with selective probes and targeting ligands to detect ROS in specific organelles or cell types of interest.

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

PubMed

Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

2017-04-01

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

Whole genomic DNA probe for detection of Porphyromonas endodontalis.

PubMed

Nissan, R; Makkar, S R; Sela, M N; Stevens, R

2000-04-01

The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled using the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphyromonas gingivalis, Porphyromonas asaccharolytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x 10(4) cells.

Probing the endocytic pathways of the filamentous bacteriophage in live cells using ratiometric pH fluorescent indicator.

PubMed

Tian, Ye; Wu, Man; Liu, Xiangxiang; Liu, Zhi; Zhou, Quan; Niu, Zhongwei; Huang, Yong

2015-02-18

Viral nanoparticles have attracted extensive research interests in diverse applications of diagnosis and therapy. In particular, filamentous M13 bacteriophages have shown great potential in biomedical applications. However, its pathways entering into cells still remain unclear, and this greatly hinders its further use as a drug or gene carrier. Here, a ratiometric M13 pH probe is designed by conjugating two fluorescent dyes onto the surface of M13. Since the intensity ratio is not influenced by probe concentration, ion strength, temperature, photobleaching, and optical path length, this ratiometric probe can be used to investigate the intracellular pH map of M13. More importantly, the internalization mechanism of M13 can be elucidated. It is found that this filamentous phage shows great cell-type dependence in interaction with cells and internalization mechanism. The phage tends to be bounded on the cell membrane of only epithelial cells, not endothelial cells. Furthermore, the M13 phage enters into cells through endocytosis with specific mechanism: clathrin-mediated endocytosis and macropinocytosis for HeLa; vesicular transport, clathrin-mediated endocytosis, and macropinocytosis for MCF-7; caveolae-mediated endocytosis for human dermal microvascular endothelial cell (HDMEC). This work provides key notes for cancer diagnosis and therapy based on filamentous bacteriophage, especially for design of pH-sensitive drug delivery systems. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanoparticle-based luminescent probes for intracellular sensing and imaging of pH.

PubMed

Schäferling, Michael

2016-05-01

Fluorescence imaging microscopy is an essential tool in biomedical research. Meanwhile, various fluorescent probes are available for the staining of cells, cell membranes, and organelles. Though, to monitor intracellular processes and dysfunctions, probes that respond to ubiquitous chemical parameters determining the cellular function such as pH, pO2 , and Ca(2+) are required. This review is focused on the progress in the design, fabrication, and application of photoluminescent nanoprobes for sensing and imaging of pH in living cells. The advantages of using nanoprobes carrying fluorescent pH indicators compared to single molecule probes are discussed as well as their limitations due to the mostly lysosomal uptake by cells. Particular attention is paid to ratiometric dual wavelength nanosensors that enable intrinsic referenced measurements. Referencing and proper calibration procedures are basic prerequisites to carry out reliable quantitative pH determinations in complex samples such as living cells. A variety of examples will be presented that highlight the diverseness of nanocarrier materials (polymers, micelles, silica, quantum dots, carbon dots, gold, photon upconversion nanocrystals, or bacteriophages), fluorescent pH indicators for the weak acidic range, and referenced sensing mechanisms, that have been applied intracellularly up to now. WIREs Nanomed Nanobiotechnol 2016, 8:378-413. doi: 10.1002/wnan.1366 For further resources related to this article, please visit the WIREs website. © 2015 Wiley Periodicals, Inc.

Identification of the glucose transporter in mammalian cell membranes using an /sup 125/(I)-forskolin photoaffinity label

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

Ruoho, A.; Wadzinski, B.; Shanahan, M.

1987-05-01

The glucose transporter has been identified in a variety of mammlian cell membranes using a carrier-free photoactivatable radioiodinated derivative of forskolin, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin, (I-125)IAPS-Fsk, at 1-10 nM. The membranes which have been photolabeled with (I-125)IAPS-Fsk are: rat cardiac sarcolemmal membranes, rat cortex and cerebellum synaptic membranes, human placental membranes, and wild type S49 lymphoma cell membranes. The glucose transporter in rat cardiac sarcolemmal membranes and rat cortex and cerebellum synaptic membranes was determined to be 45 kDa by SDS-PAGE. Photolysis of human placental membranes and S49 lymphoma membranes with (I-125)IAPS-Fsk followed by SDS-PAGE indicated specific derivatization of a broad band (45-55more » kDa) in placental membranes and a narrower band (45 kDa) in the S49 lymphoma membranes. Digestion of the (I-125)IPAS-Fsk labelled placental and S49 lymphoma membranes with endo-B-galactosidase showed a reduction in the apparent molecular weight of the radiolabelled band to 40 kDa. Trypsinization of labelled placental and lymphoma membranes produced an 18 kDa radiolabelled proteolytic fragment. (I-125)IAPS-Fsk is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.« less

Mitochondrial ryanodine-sensitive Ca2+ channels of rat liver.

PubMed

Kupynyak, N I; Ikkert, O V; Shlykov, S G; Babich, L G; Manko, V V

2017-01-01

To examine ryanodine-sensitive Ca 2+ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca 2+ concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca 2+ concentration and affecting the energy state of mictochondria in hepatocytes. Copyright © 2017 John Wiley & Sons, Ltd.

Molecular diversity of mesophilic and thermophilic bacteria in a membrane bioreactor determined by fluorescent in situ hybridization with mxaF- and rRNA-targeted probes.

PubMed

Dias, João Carlos T; Silva, Cláudio M; Mounteer, Ann H; Passos, Flavia M L; Linardi, Valter R

2003-01-01

An evaluation of the efficiency of treatment of kraft mill foul condensates in a membrane bioreactor was carried out in the laboratory. Efficiency and rate of methanol removal were quantified at operating temperatures of 35, 45 and 55 degrees C. The structure of the bacterial community present in the reactor biomass at the different operating temperatures was evaluated by in situ hybridization of the biomass samples with fluorescently-labelled probes (FISH) targeting the Eubacteria, the alpha, beta and gamma subclasses of the Proteobacteria, the low G + C content Gram-positive bacteria (Bacillus spp.), while community function was evaluated by in situ hybridization with a methanol dehydrogenase gene (mxaF) probe. Methanol removal efficiency decreased from 99.4 to 92%, and removal rate from 2.69 mg MeOH/l x min to 2.49 mg MeOH/l x min when the operating temperature was increased from 35 to 55 degrees C. This decrease in methanol removal was accompanied by a decrease (from 58% to 42%) in the relative proportion of cells that hybridized with the mxaF probe. The relative proportion of Bacillus spp. increased from 5 to 20% while the proportion of members of the alpha subclass of Proteobacteria decreased from 16% to 6% when the bioreactor operating temperature was raised from 35 to 55 degrees C. The relative proportions of bacteria belonging to the beta (22-25%) and gamma (18-20%) subclasses of the Proteobacteria remained relatively constant regardless of operating temperature. Proteobacteria (alpha, beta and gamma subclasses) and Bacillus spp. represented 61, 67 and 71% of the Eubacteria in the biomass sampled at 35, 45 and 55 degrees C, respectively. The FISH technique was shown to be an efficient method for detection of both structural and functional changes in the bacterial communities that could be related to efficiency of methanol removal in a membrane bioreactor operating at different temperatures.

All-optical modulation in Mid-Wavelength Infrared using porous Si membranes

PubMed Central

Park, Sung Jin; Zakar, Ammar; Zerova, Vera L.; Chekulaev, Dimitri; Canham, Leigh T.; Kaplan, Andre

2016-01-01

We demonstrate for the first time the possibility of all-optical modulation of self-standing porous Silicon (pSi) membrane in the Mid-Wavelength Infrared (MWIR) range using femtosecond pump-probe techniques. To study optical modulation, we used pulses of an 800 nm, 60 femtosecond for pump and a MWIR tunable probe in the spectral range between 3.5 and 4.4 μm. We show that pSi possesses a natural transparency window centred around 4 μm. Yet, about 55% of modulation contrast can be achieved by means of optical excitation at the pump power of 60 mW (4.8 mJ/cm2). Our analysis shows that the main mechanism of the modulation is interaction of the MWIR signal with the free charge carrier excited by the pump. The time-resolved measurements showed a sub-picosecond rise time and a recovery time of about 66 ps, which suggests a modulation speed performance of ~15 GHz. This optical modulation of pSi membrane in MWIR can be applied to a variety of applications such as thermal imaging and free space communications. PMID:27440224

Temperature-induced changes in lecithin model membranes detected by novel covalent spin-labelled phospholipids.

PubMed

Stuhne-Sekalec, L; Stanacev, N Z

1977-02-01

Several spin-labelled phospholipids carrying covalently bound 5-doxylstearic acid (2-(3-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl) were intercalated in liposomes of saturated and unsaturated lecithins. Temperature-induced changes of these liposomes, detected by the spin-labelled phospholipids, were found to be in agreement with the previously described transitions of hydrocarbon chains of host lecithins detected by different probes and different techniques, establishing that spin-labelled phosopholipids are sensitive probes for the detection of temperature-induced changes in lecithin model membranes. In addition to the detection of already-known transitions in lecithin liposomes, the coexistence of two distinctly different enviroments was observed above the characteristic transition temperature. This phenomenon was tentatively attributed to the influence of the lecithin polar group on the fluidity of fatty acyl chains near the polar group. Combined with other results from the literature, the coexistence of two environments could be associated with the coexistence of two conformational isomers of lecithin, differing in the orientation of the polar head group with respect to the plane of bilayer. These findings have been discussed in view of the present state of knowledge regarding temperature-induced changes in model membranes.

Biotinylated vanadium and chromium sulfide nanoparticles as probes for colocalization of membrane proteins.

PubMed

Loukanov, Alexandre; Emin, Saim

2016-09-01

We report the microemulsion synthesis of vanadium and chromium sulfide nanoparticles (NPs) and their biological application as nanoprobes for colocalization of membrane proteins. Spherical V2 S3 and Cr2 S3 NPs were prepared in reverse microemulsion droplets, as nanoreactors, obtained by the surfactant sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in nonpolar organic phase (heptane). Electron microscopic data indicated that the size distribution of the nanoparticles was uniform with an average diameter between 3 ÷ 5 nm. The prepared hydrophobic nanocrystals were transferred in aqueous phase by surface cap exchange of AOT with biotin-dihydrolipoic ligands. This substitution allows the nanoparticles solubility in aqueous solutions and confer their bioactivity. In addition, we report the conjugation procedure between α-Lipoic acid (LA) and biotin (abbreviated as biotin-LA). The biotin-LA structure was characterized by 1D and 2D NMR spectroscopy. The biotinylated vanadium and chromium sulfide nanoparticles were tested as probes for colocalization of glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. The method suggests their high labeling efficiency for study of membrane biological macromolecules. Microsc. Res. Tech. 79:799-805, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

PubMed

Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-03-01

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif

PubMed Central

Wang, Yaofeng; Kraut, Rachel; Mu, Yuguang

2015-01-01

The Amyloid-β (Aβ)-derived, sphingolipid binding domain (SBD) peptide is a fluorescently tagged probe used to trace the diffusion behavior of sphingolipid-containing microdomains in cell membranes through binding to a constellation of glycosphingolipids, sphingomyelin, and cholesterol. However, the molecular details of the binding mechanism between SBD and plasma membrane domains remain unclear. Here, to investigate how the peptide recognizes the lipid surface at an atomically detailed level, SBD peptides in the environment of raft-like bilayers were examined in micro-seconds-long molecular dynamics simulations. We found that SBD adopted a coil-helix-coil structural motif, which binds to multiple GT1b gangliosides via salt bridges and CH–π interactions. Our simulation results demonstrate that the CH–π and electrostatic forces between SBD monomers and GT1b gangliosides clusters are the main driving forces in the binding process. The presence of the fluorescent dye and linker molecules do not change the binding mechanism of SBD probes with gangliosides, which involves the helix-turn-helix structural motif that was suggested to constitute a glycolipid binding domain common to some sphingolipid interacting proteins, including HIV gp120, prion, and Aβ. PMID:26540054

A subsequent closed-form description of propagated signaling phenomena in the membrane of an axon

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

Melendy, Robert F., E-mail: rfmelendy@liberty.edu

2016-05-15

I recently introduced a closed-form description of propagated signaling phenomena in the membrane of an axon [R.F. Melendy, Journal of Applied Physics 118, 244701 (2015)]. Those results demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation, function together in generating an action potential in a unified, closed-form description. At present, I report on a subsequent closed-form model that unifies intracellular conductance and the thermodynamics of magnetization, with the membrane electric field, E{sub m}. It’s anticipated this work will compel researchers in biophysics, physical biology, and the computational neurosciences, to probe deeper into the classical and quantum features ofmore » membrane magnetization and signaling, informed by the computational features of this subsequent model.« less

Tensiometer methods and apparatus

DOEpatents

Grover, Blair K.; Hubbell, Joel M.; Sisson, James B.; Casper, William L.

2004-08-10

A method for collecting data regarding a matric potential of a media includes providing a tensiometer having a stainless steel tensiometer casing, the stainless steel tensiometer casing comprising a tip portion which includes a wetted porous stainless steel membrane through which a matric potential of a media is sensed; driving the tensiometer into the media using an insertion tube comprising a plurality of probe casing which are selectively coupled to form the insertion tube as the tensiometer is progressively driven deeper into the media, wherein the wetted porous stainless steel membrane is in contact with the media; and sensing the matric potential the media exerts on the wetted porous stainless steel membrane by a pressure sensor in fluid hydraulic connection with the porous stainless steel membrane. A tensiometer includes a stainless steel casing.

Probe Into the Influence of Crosslinking on CO2 Permeation of Membranes

PubMed Central

Li, Jinghui; Chen, Zhuo; Umar, Ahmad; Liu, Yang; Shang, Ying; Zhang, Xiaokai; Wang, Yao

2017-01-01

Crosslinking is an effective way to fabricate high-selective CO2 separation membranes because of its unique crosslinking framework. Thus, it is essentially significant to study the influence of crosslinking degree on the permeation selectivities of CO2. Herein, we report a successful and facile synthesis of a series of polyethylene oxide (PEO)-based diblock copolymers (BCP) incorporated with an unique UV-crosslinkable chalcone unit using Reversible Addition-Fragmentation Chain Transfer Polymerization (RAFT) process. The membranes of as-prepared BCPs show superior carbon dioxide (CO2) separation properties as compared to nitrogen (N2) after UV-crosslinking. Importantly, the influence of different proportions of crosslinked chalcone on CO2 selectivities was systematically investigated, which revealed that CO2 selectivities increased obviously with the enhancement of chalcone fractions within a certain limit. Further, the CO2 selectivities of block copolymer with the best block proportion was studied by varying the crosslinking time which confirmed that the high crosslinking degree exhibited a better CO2/N2 (αCO2/N2) selectivities. A possible mechanism model revealing that the crosslinking degree played a key role in the gas separation process was also proposed. PMID:28051190

Probe Into the Influence of Crosslinking on CO2 Permeation of Membranes

NASA Astrophysics Data System (ADS)

Li, Jinghui; Chen, Zhuo; Umar, Ahmad; Liu, Yang; Shang, Ying; Zhang, Xiaokai; Wang, Yao

2017-01-01

Crosslinking is an effective way to fabricate high-selective CO2 separation membranes because of its unique crosslinking framework. Thus, it is essentially significant to study the influence of crosslinking degree on the permeation selectivities of CO2. Herein, we report a successful and facile synthesis of a series of polyethylene oxide (PEO)-based diblock copolymers (BCP) incorporated with an unique UV-crosslinkable chalcone unit using Reversible Addition-Fragmentation Chain Transfer Polymerization (RAFT) process. The membranes of as-prepared BCPs show superior carbon dioxide (CO2) separation properties as compared to nitrogen (N2) after UV-crosslinking. Importantly, the influence of different proportions of crosslinked chalcone on CO2 selectivities was systematically investigated, which revealed that CO2 selectivities increased obviously with the enhancement of chalcone fractions within a certain limit. Further, the CO2 selectivities of block copolymer with the best block proportion was studied by varying the crosslinking time which confirmed that the high crosslinking degree exhibited a better CO2/N2 (αCO2/N2) selectivities. A possible mechanism model revealing that the crosslinking degree played a key role in the gas separation process was also proposed.

Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly.

PubMed

O'Neill, Sharon; Mathis, Magalie; Kovačič, Lidija; Zhang, Suisheng; Reinhardt, Jürgen; Scholz, Dimitri; Schopfer, Ulrich; Bouhelal, Rochdi; Knaus, Ulla G

2018-06-08

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22 phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22 phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22 phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Membrane fluidity controls redox-regulated cold stress responses in cyanobacteria.

PubMed

Maksimov, Eugene G; Mironov, Kirill S; Trofimova, Marina S; Nechaeva, Natalya L; Todorenko, Daria A; Klementiev, Konstantin E; Tsoraev, Georgy V; Tyutyaev, Eugene V; Zorina, Anna A; Feduraev, Pavel V; Allakhverdiev, Suleyman I; Paschenko, Vladimir Z; Los, Dmitry A

2017-09-01

Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.

Peptide-membrane Interactions by Spin-labeling EPR

PubMed Central

Smirnova, Tatyana I.; Smirnov, Alex I.

2016-01-01

Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described. PMID:26477253

Evaluation of the membrane lipid selectivity of the pea defensin Psd1.

PubMed

Gonçalves, Sónia; Teixeira, Alexandre; Abade, João; de Medeiros, Luciano Neves; Kurtenbach, Eleonora; Santos, Nuno C

2012-05-01

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent. Copyright © 2012 Elsevier B.V. All rights reserved.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor cell invasion through the basement membrane in C. elegans

PubMed Central

Kelley, Laura C.; Wang, Zheng; Hagedorn, Elliott J.; Wang, Lin; Shen, Wanqing; Lei, Shijun; Johnson, Sam A.; Sherwood, David R.

2018-01-01

Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory. PMID:28880279

The graphene oxide membrane immersing in the aqueous solution studied by electrochemical impedance spectroscopy

NASA Astrophysics Data System (ADS)

Zhang, Yongjing; Chen, Zhe; Yao, Lei; Wang, Xiao; Fu, Ping; Lin, Zhidong

2018-04-01

The interlayer spacing of graphene oxide (GO) is a key property for GO membrane. To probe the variation of interlayer spacing of the GO membrane immersing in KCl aqueous solution, electrochemical impedance spectroscopy (EIS), x-ray diffraction (XRD) and computational calculation was utilized in this study. The XRD patterns show that soaking in KCl aqueous solution leads to an increase of interlayer spacing of GO membrane. And the EIS results indicate that during the immersing process, the charge transfer resistance of GO membrane decreases first and then increases. Computational calculation confirms that intercalated water molecules can result in an increase of interlayer spacing of GO membrane, while the permeation of K+ ions would lead to a decrease of interlayer spacing. All the results are in agreement with each other. It suggests that during the immersing process, the interlayer spacing of GO enlarges first and then decreases. EIS can be a promisingly online method for examining the interlayer spacing of GO in the aqueous solution.

Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

PubMed Central

Székács, Inna; Kaszás, Nóra; Gróf, Pál; Erdélyi, Katalin; Szendrő, István; Mihalik, Balázs; Pataki, Ágnes; Antoni, Ferenc A.; Madarász, Emilia

2013-01-01

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na+ and organic cations through gramicidin channels and detecting the Cl–-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline. PMID:24339925

Preparation of PVA membrane for immobilization of GOD for glucose biosensor.

PubMed

Kumar, Jitendra; D'Souza, S F

2008-03-15

A membrane was prepared using polyvinyl alcohol (PVA) with low and high degree of polymerization (DOP), acetone, benzoic acid (BA) and was cross-linked by UV treatment. Membrane composition was optimized on the basis of swelling index. Membrane prepared with 12% low DOP and 8% high DOP of PVA, 2% BA, dissolved in buffer containing 20% acetone and cross-linked with UV treatment exhibited lower swelling index. Fourier transform infrared (FTIR) study of the membranes showed appearance of a strong band at approximately 2337 cm(-1) when UV was used for cross-linking in the presence of benzoic acid. Scanning electron microscope (SEM) study revealed that membrane cross-linked with UV treatment was smoother. Glucose oxidase (GOD)-PVA membrane was associated with the dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose to gluconolactone. A wide detection range, 0.9-225 mg/dl was estimated from the linear range of calibration plot of biosensor reading. Membranes were reused for 32 reactions without significant loss of activity and stored for 30 days (approximately 90% activity) at 4 degrees C. Membranes were also used with real blood samples.

Methods to Assess the Direct Interaction of C. jejuni with Mucins.

PubMed

Clyne, Marguerite; Duggan, Gina; Naughton, Julie; Bourke, Billy

2017-01-01

Studies of the interaction of bacteria with mucus-secreting cells can be complemented at a more mechanistic level by exploring the interaction of bacteria with purified mucins. Here we describe a far Western blotting approach to show how C. jejuni proteins separated by SDS PAGE and transferred to a membrane or slot blotted directly onto a membrane can be probed using biotinylated mucin. In addition we describe the use of novel mucin microarrays to assess bacterial interactions with mucins in a high-throughput manner.

Determination of Novel Strategies for Hastening Corneal Wound Healing and Reducing Tissue Inflammation

DTIC Science & Technology

2011-10-01

blocking buffer, 5% fat -free milk in 0.1% Tris-buffered solution/Tween-20, for 1 hour at room temperature and then probed overnight at 5°C with...and blotting onto Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA). Membranes were blocked with blocking buffer, 5% fat -free milk in 1x PBS buffer...distribution unlimited 13. SUPPLEMENTARY NOTES The aim of this study is to uncover novel transient receptor potential protein vanilloid-1 (TRPV1

Collagen membranes: a review.

PubMed

Bunyaratavej, P; Wang, H L

2001-02-01

Collagen materials have been utilized in medicine and dentistry because of their proven biocompatability and capability of promoting wound healing. For guided tissue regeneration (GTR) procedures, collagen membranes have been shown to be comparable to non-absorbable membranes with regard to probing depth reduction, clinical attachment gain, and percent of bone fill. Although these membranes are absorbable, collagen membranes have been demonstrated to prevent epithelial down-growth along the root surfaces during the early phase of wound healing. The use of grafting material in combination with collagen membranes seems to improve clinical outcomes for furcation, but not intrabony, defects when compared to the use of membranes alone. Recently, collagen materials have also been applied in guided bone regeneration (GBR) and root coverage procedures with comparable success rates to non-absorbable expanded polytetrafluoroethylene (ePTFE) membranes and conventional subepithelial connective tissue grafts, respectively. Long-term clinical trials are still needed to further evaluate the benefits of collagen membranes in periodontal and peri-implant defects. This article will review the rationale for each indication and its related literature, both in vitro and in vivo studies. The properties that make collagen membranes attractive for use in regenerative therapy will be addressed. In addition, varieties of cross-linking techniques utilized to retard the degradation rate of collagen membranes will be discussed.

Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. Axial bright-field STEM tomography.

PubMed

McBride, E L; Rao, A; Zhang, G; Hoyne, J D; Calco, G N; Kuo, B C; He, Q; Prince, A A; Pokrovskaya, I D; Storrie, B; Sousa, A A; Aronova, M A; Leapman, R D

2018-06-01

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach. Copyright © 2018. Published by Elsevier Inc.

Scanning probe microscopy of biomedical interfaces

NASA Astrophysics Data System (ADS)

Vansteenkiste, S. O.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.

1998-02-01

The development of the scanning probe microscopes over the past decade has provided a number of exciting new surface analytical techniques making a significant progress in the characterisation of biomedical interfaces. In this review, several examples are presented to illustrate that SPM is a powerful and promising tool for surface investigations including biomolecules, cell membranes, polymers and even living cells. The ability of the SPM instrument to monitor adhesion phenomena and provide quantitative information about intermolecular interactions is also described. Moreover, the huge potential of the scanning probe microscopes to study dynamic processes at interfaces under nearly physiological conditions is highlighted. Novel applications in the field of biochemistry, microbiology, biomaterial engineering, drug delivery and even medicine are discussed.

Optical Imaging of Mammary and Prostate Tumors in Living Animals using a Synthetic Near Infrared Zinc(II)-Dipicolylamine Probe for Anionic Cell Surfaces

PubMed Central

Smith, Bryan A.; Akers, Walter J.; Leevy, W. Matthew; Lampkins, Andrew J.; Xiao, Shuzhang; Wolter, William; Suckow, Mark A.; Achilefu, Samuel; Smith, Bradley D.

2009-01-01

In vivo optical imaging shows that a fluorescent imaging probe, comprised of a near-infrared fluorophore attached to an affinity group containing two zinc(II)-dipicolylamine (Zn-DPA) units, targets prostate and mammary tumors in two different xenograft animal models. The tumor selectivity is absent with control fluorophores whose structures do not have appended Zn-DPA targeting ligands. Ex vivo biodistribution and histological analyses indicate that the probe is targeting the necrotic regions of the tumors, which is consistent with in vitro microscopy showing selective targeting of the anionic membrane surfaces of dead and dying cells. PMID:20014845

Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

DOEpatents

Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

1992-01-01

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

DOEpatents

Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

1993-01-01

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.

PubMed

Nodzyński, Tomasz; Vanneste, Steffen; Zwiewka, Marta; Pernisová, Markéta; Hejátko, Jan; Friml, Jiří

2016-11-07

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

PubMed

Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

2016-01-26

Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application.

Cellular imaging using BODIPY-, pyrene- and phthalocyanine-based conjugates.

PubMed

Bizet, Faustine; Ipuy, Martin; Bernhard, Yann; Lioret, Vivian; Winckler, Pascale; Goze, Christine; Perrier-Cornet, Jean-Marie; Decréau, Richard A

2018-01-15

Fluorescent Probes aimed at absorbing in the blue/green region of the spectrum and emitting in the green/red have been synthesized (as the form of dyads-pentads), studied by spectrofluorimetry, and used for cellular imaging. The synthesis of phthalocyanine-pyrene 1 was achieved by cyclotetramerization of pyrenyldicyanobenzene, whereas phthalocyanine-BODIPY 2c was synthesized by Sonogashira coupling between tetraiodophthalocyanine and meso-alkynylBODIPY. The standard four-steps BODIPY synthesis was applied to the BODIPY-pyrene dyad 3 starting from pyrenecarbaldehyde and dimethylpyrrole. 1 H, 13 C, 19 F, 11 BNMR, ICP, MS, and UV/Vis spectroscopic analyses demonstrated that 2c is a mixture of BODIPY-Pc conjugates corresponding to an average ratio of 2.5 BODIPY per Pc unit, where its bis, tris, tetrakis components could not be separated. Fluorescence emission studies (μM concentration in THF) showed that the design of the probes allowed excitation of their antenna (pyrene, BODIPY) in the blue/green region of the spectrum, and subsequent transfer to the acceptor platform (BODIPY, phthalocyanine) followed by its emission in the green/red (with up to 140-350 nm overall Stokes shifts). The fluorescent probes were used for cellular imaging of B16F10 melanoma cells upon solubilization in 1% DMSO containing RPMI or upon encapsulation in liposomes (injection method). Probes were used at 1-10 μM concentrations, cells were fixed with methanol and imaged by biphoton and/or confocal microscopy, showing that probes could achieve the staining of cells membranes and not the nucleus. Copyright © 2017. Published by Elsevier Ltd.

Selective chromo-fluorogenic detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) with a unique probe based on a boron dipyrromethene (BODIPY) dye.

PubMed

Barba-Bon, Andrea; Costero, Ana M; Gil, Salvador; Martínez-Máñez, Ramón; Sancenón, Félix

2014-11-21

A novel colorimetric probe (P4) for the selective differential detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) was prepared. Probe P4 contains three reactive sites; i.e. (i) a nucleophilic phenol group able to undergo phosphorylation with nerve gases, (ii) a carbonyl group as a reactive site for cyanide; and (iii) a triisopropylsilyl (TIPS) protecting group that is known to react with fluoride. The reaction of P4 with DCNP in acetonitrile resulted in both the phosphorylation of the phenoxy group and the release of cyanide, which was able to react with the carbonyl group of P4 to produce a colour modulation from pink to orange. In contrast, phosphorylation of P4 with DFP in acetonitrile released fluoride that hydrolysed the TIPS group in P4 to yield a colour change from pink to blue. Probe P4 was able to discriminate between DFP and DCNP with remarkable sensitivity; limits of detection of 0.36 and 0.40 ppm for DCNP and DFP, respectively, were calculated. Besides, no interference from other organophosphorous derivatives or with presence of acid was observed. The sensing behaviour of P4 was also retained when incorporated into silica gel plates or onto polyethylene oxide membranes, which allowed the development of simple test strips for the colorimetric detection of DCNP and DFP in the vapour phase. P4 is the first probe capable of colorimetrically differentiating between a Tabun mimic (DCNP) and a Sarin and Soman mimic (DFP).

Effect of Plasma Membrane Semipermeability in Making the Membrane Electric Double Layer Capacitances Significant.

PubMed

Sinha, Shayandev; Sachar, Harnoor Singh; Das, Siddhartha

2018-01-30

Electric double layers (or EDLs) formed at the membrane-electrolyte interface (MEI) and membrane-cytosol interface (MCI) of a charged lipid bilayer plasma membrane develop finitely large capacitances. However, these EDL capacitances are often much larger than the intrinsic capacitance of the membrane, and all of these capacitances are in series. Consequently, the effect of these EDL capacitances in dictating the overall membrane-EDL effective capacitance C eff becomes negligible. In this paper, we challenge this conventional notion pertaining to the membrane-EDL capacitances. We demonstrate that, on the basis of the system parameters, the EDL capacitance for both the permeable and semipermeable membranes can be small enough to influence C eff . For the semipermeable membranes, however, this lowering of the EDL capacitance can be much larger, ensuring a reduction of C eff by more than 20-25%. Furthermore, for the semipermeable membranes, the reduction in C eff is witnessed over a much larger range of system parameters. We attribute such an occurrence to the highly nonintuitive electrostatic potential distribution associated with the recently discovered phenomena of charge-inversion-like electrostatics and the attainment of a positive zeta potential at the MCI for charged semipermeable membranes. We anticipate that our findings will impact the quantification and the identification of a large number of biophysical phenomena that are probed by measuring the plasma membrane capacitance.

Microbubble-Assisted Ultrasound-Induced Transient Phosphatidylserine Translocation.

PubMed

Escoffre, Jean-Michel; Derieppe, Marc; Lammertink, Bart; Bos, Clemens; Moonen, Chrit

2017-04-01

Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells. Sonopermeabilization induced transient membrane permeabilization, which is positively correlated with reversible PS externalization. This membrane disorganization was temporary and not associated with loss of cell viability. Sonopermeabilization did not induce PS externalization via activation of the scramblase. We hypothesize that acoustically induced membrane pores may provide a new pathway for PS migration between both membrane leaflets. During the membrane-resealing phase, PS asymmetry may be re-established by amino-phospholipid flippase activity and/or endocytosis, along with exocytosis processes. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

In situ probing the interior of single bacterial cells at nanometer scale

NASA Astrophysics Data System (ADS)

Liu, Boyin; Hemayet Uddin, Md; Ng, Tuck Wah; Paterson, David L.; Velkov, Tony; Li, Jian; Fu, Jing

2014-10-01

We report a novel approach to probe the interior of single bacterial cells at nanometre resolution by combining focused ion beam (FIB) and atomic force microscopy (AFM). After removing layers of pre-defined thickness in the order of 100 nm on the target bacterial cells with FIB milling, AFM of different modes can be employed to probe the cellular interior under both ambient and aqueous environments. Our initial investigations focused on the surface topology induced by FIB milling and the hydration effects on AFM measurements, followed by assessment of the sample protocols. With fine-tuning of the process parameters, in situ AFM probing beneath the bacterial cell wall was achieved for the first time. We further demonstrate the proposed method by performing a spatial mapping of intracellular elasticity and chemistry of the multi-drug resistant strain Klebsiella pneumoniae cells prior to and after it was exposed to the ‘last-line’ antibiotic polymyxin B. Our results revealed increased stiffness occurring in both surface and interior regions of the treated cells, suggesting loss of integrity of the outer membrane from polymyxin treatments. In addition, the hydrophobicity measurement using a functionalized AFM tip was able to highlight the evident hydrophobic portion of the cell such as the regions containing cell membrane. We expect that the proposed FIB-AFM platform will help in gaining deeper insights of bacteria-drug interactions to develop potential strategies for combating multi-drug resistance.

Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

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

Thomson, M.S.

1988-01-01

Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture andmore » used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two {sup 32}P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos.« less

Tethered Hsp90 Inhibitors Carrying Optical or Radioiodinated Probes Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

PubMed Central

Barrott, Jared J.; Hughes, Philip F.; Osada, Takuya; Yang, Xiao-Yi; Hartman, Zachary C.; Loiselle, David R.; Spector, Neil L.; Neckers, Len; Rajaram, Narasimhan; Hu, Fangyao; Ramanujam, Nimmi; Vaidyanathan, Ganesan; Zalutsky, Michael R.; Lyerly, H. Kim; Haystead, Timothy A.

2013-01-01

Summary Hsp90 inhibitors have demonstrated unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained but could be influenced by ectopically expressed Hsp90 in tumors. We have synthesized novel Hsp90 inhibitors that can carry optical or radioiodinated probes via a PEG tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein is occurring at the plasma membrane. In mice, we show exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors targeting ectopic Hsp90 can be used to detect breast cancer malignancies through non-invasive imaging. PMID:24035283

Non-radioactive detection of trinucleotide repeat size variability.

PubMed

Tomé, Stéphanie; Nicole, Annie; Gomes-Pereira, Mario; Gourdon, Genevieve

2014-03-06

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

PubMed Central

Marriott, G.; Heidecker, M.; Diamandis, E. P.; Yan-Marriott, Y.

1994-01-01

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7811952

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

Cheng, Chi-Yuan; Kausik, Ravinath; Han, Songi, E-mail: songi@chem.ucsb.edu

Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed {sup 1}H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5–10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroupsmore » or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster surface water diffusivity. Another is the concurrent tightening of lipid packing that reduces passive, possibly unwanted, diffusion of ions and water across the bilayer.« less

Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

PubMed

Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

2014-04-01

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

Effect of CO2 absorption on ion and water mobility in an anion exchange membrane

NASA Astrophysics Data System (ADS)

Peng, Jing; Roy, Asa L.; Greenbaum, Steve G.; Zawodzinski, Thomas A.

2018-03-01

We report the measured water uptake, density, ionic conductivity and water transport properties in Tokuyama A201 membrane in OH-, HCO3- and Cl- forms. The water uptake of the AEM varies with anion type in the order λ(OH-) > λ(HCO3-) > λ(Cl-) for samples equilibrated with the same water vapor activity (aw). The conductivity of the AEM is reduced by absorption of CO2. Pulsed-field gradient nuclear magnetic resonance (PFG-NMR) measurements were utilized to characterize the diffusivity of water and HCO3- ion. The anion diffusion coefficient and membrane conductivity are used to probe the applicability of the Nernst-Einstein equation in these AEMs.

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells.

PubMed

Wüstner, Daniel; Lund, Frederik W; Röhrl, Clemens; Stangl, Herbert

2016-01-01

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

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

Cameron, R.S.; Cameron, P.L.; Castle, J.D.

1986-10-01

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pImore » and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.« less

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

PubMed

van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

2014-09-01

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

Integrating nanohybrid membranes of reduced graphene oxide: chitosan: silica sol gel with fiber optic SPR for caffeine detection

NASA Astrophysics Data System (ADS)

Kant, Ravi; Tabassum, Rana; Gupta, Banshi D.

2017-05-01

Caffeine is the most popular psychoactive drug consumed in the world for improving alertness and enhancing wakefulness. However, caffeine consumption beyond limits can result in lot of physiological complications in human beings. In this work, we report a novel detection scheme for caffeine integrating nanohybrid membranes of reduced graphene oxide (rGO) in chitosan modified silica sol gel (rGO: chitosan: silica sol gel) with fiber optic surface plasmon resonance. The chemically synthesized nanohybrid membrane forming the sensing route has been dip coated over silver coated unclad central portion of an optical fiber. The sensor works on the mechanism of modification of dielectric function of sensing layer on exposure to analyte solution which is manifested in terms of red shift in resonance wavelength. The concentration of rGO in polymer network of chitosan and silica sol gel and dipping time of the silver coated probe in the solution of nanohybrid membrane have been optimized to extricate the supreme performance of the sensor. The optimized sensing probe possesses a reasonably good sensitivity and follows an exponentially declining trend within the entire investigating range of caffeine concentration. The sensor boasts of an unparalleled limit of detection value of 1.994 nM and works well in concentration range of 0-500 nM with a response time of 16 s. The impeccable sensor methodology adopted in this work combining fiber optic SPR with nanotechnology furnishes a novel perspective for caffeine determination in commercial foodstuffs and biological fluids.

Probing effects of pressure release on virus capture during virus filtration using confocal microscopy.

PubMed

Dishari, Shudipto K; Venkiteshwaran, Adith; Zydney, Andrew L

2015-10-01

Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes using bacteriophage φx174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve® NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve® Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure. © 2015 Wiley Periodicals, Inc.

Confocal Raman Microscopy for In-situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles

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

Kitt, Jay P.; Bryce, David A.; Minteer, Shelley D.

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this paper, we employ in-situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayersmore » deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically-trapped phospholipid vesicle membranes. Finally and additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.« less

Confocal Raman Microscopy for In-situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles

DOE PAGES

Kitt, Jay P.; Bryce, David A.; Minteer, Shelley D.; ...

2018-05-14

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this paper, we employ in-situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayersmore » deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically-trapped phospholipid vesicle membranes. Finally and additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.« less

Confocal Raman Microscopy for in Situ Measurement of Phospholipid-Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles.

PubMed

Kitt, Jay P; Bryce, David A; Minteer, Shelley D; Harris, Joel M

2018-06-05

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this work, we employ in situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayers deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically trapped phospholipid vesicle membranes. Additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.

In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives.

PubMed

Jiang, Yao-Wen; Guo, Hao-Yue; Chen, Zhan; Yu, Zhi-Wu; Wang, Zhifei; Wu, Fu-Gen

2016-07-05

Lipid rafts are highly ordered small microdomains mainly composed of glycosphingolipids, cholesterol, and protein receptors. Optically distinguishing lipid raft domains in cell membranes would greatly facilitate the investigations on the structure and dynamics of raft-related cellular behaviors, such as signal transduction, membrane transport (endocytosis), adhesion, and motility. However, current strategies about the visualization of lipid raft domains usually suffer from the low biocompatibility of the probes, invasive detection, or ex situ observation. At the same time, naturally derived biomacromolecules have been extensively used in biomedical field and their interaction with cells remains a long-standing topic since it is closely related to various fundamental studies and potential applications. Herein, noninvasive visualization of lipid raft domains in model lipid bilayers (supported lipid bilayers and giant unilamellar vesicles) and live cells was successfully realized in situ using fluorescent biomacromolecules: the fluorescein isothiocyanate (FITC)-labeled glycol chitosan molecules. We found that the lipid raft domains in model or real membranes could be specifically stained by the FITC-labeled glycol chitosan molecules, which could be attributed to the electrostatic attractive interaction and/or hydrophobic interaction between the probes and the lipid raft domains. Since the FITC-labeled glycol chitosan molecules do not need to completely insert into the lipid bilayer and will not disturb the organization of lipids, they can more accurately visualize the raft domains as compared with other fluorescent dyes that need to be premixed with the various lipid molecules prior to the fabrication of model membranes. Furthermore, the FITC-labeled glycol chitosan molecules were found to be able to resist cellular internalization and could successfully visualize rafts in live cells. The present work provides a new way to achieve the imaging of lipid rafts and also sheds new light on the interaction between biomacromolecules and lipid membranes.

Rapid ultrasensitive single particle surface-enhanced Raman spectroscopy using metallic nanopores.

PubMed

Cecchini, Michael P; Wiener, Aeneas; Turek, Vladimir A; Chon, Hyangh; Lee, Sangyeop; Ivanov, Aleksandar P; McComb, David W; Choo, Jaebum; Albrecht, Tim; Maier, Stefan A; Edel, Joshua B

2013-10-09

Nanopore sensors embedded within thin dielectric membranes have been gaining significant interest due to their single molecule sensitivity and compatibility of detecting a large range of analytes, from DNA and proteins, to small molecules and particles. Building on this concept we utilize a metallic Au solid-state membrane to translocate and rapidly detect single Au nanoparticles (NPs) functionalized with 589 dye molecules using surface-enhanced resonance Raman spectroscopy (SERRS). We show that, due to the plasmonic coupling between the Au metallic nanopore surface and the NP, signal intensities are enhanced when probing analyte molecules bound to the NP surface. Although not single molecule, this nanopore sensing scheme benefits from the ability of SERRS to provide rich vibrational information on the analyte, improving on current nanopore-based electrical and optical detection techniques. We show that the full vibrational spectrum of the analyte can be detected with ultrahigh spectral sensitivity and a rapid temporal resolution of 880 μs.

Molecular dynamics simulations of lysozyme-lipid systems: probing the early steps of protein aggregation.

PubMed

Trusova, Valeriya M; Gorbenko, Galyna P

2017-07-10

Using the molecular dynamics simulation, the role of lipids in the lysozyme transition into the aggregation-competent conformation has been clarified. Analysis of the changes of lysozyme secondary structure upon its interactions with the model bilayer membranes composed of phosphatidylcholine and its mixtures with phosphatidylglycerol (10, 40, and 80 mol%) within the time interval of 100 ns showed that lipid-bound protein is characterized by the increased content of β-structures. Along with this, the formation of protein-lipid complexes was accompanied by the increase in the gyration radius and the decrease in RMSD of polypeptide chain. The results obtained were interpreted in terms of the partial unfolding of lysozyme molecule on the lipid matrix, with the magnitude of this effect being increased with increasing the fraction of anionic lipids. Based on the results of molecular dynamics simulation, a hypothetical model of the nucleation of lysozyme amyloid fibrils in a membrane environment was suggested.

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers

DOE PAGES

Carrillo, Jan-Michael Y.; Katsaras, John; Sumpter, Bobby G.; ...

2017-01-12

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to signficant insights into vital membrane phenomena such as domain formation, passive permeation and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatially and temporally. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular under- standing of lipid membranes. Particularly, the synergymore » between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. We also describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidity. Specfically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for investigations of more complex membrane systems.« less

Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update.

PubMed

Wolff, Antonio C; Hammond, M Elizabeth Hale; Allison, Kimberly H; Harvey, Brittany E; Mangu, Pamela B; Bartlett, John M S; Bilous, Michael; Ellis, Ian O; Fitzgibbons, Patrick; Hanna, Wedad; Jenkins, Robert B; Press, Michael F; Spears, Patricia A; Vance, Gail H; Viale, Giuseppe; McShane, Lisa M; Dowsett, Mitchell

2018-05-30

Purpose To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. Methods Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. Recommendations Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in > 10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended work-up for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥ 2.0; average HER2 copy number < 4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 4.0 and < 6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results. Find additional information at www.asco.org/breast-cancer-guidelines .

Protein Diffusion on Charged Membranes: A Dynamic Mean-Field Model Describes Time Evolution and Lipid Reorganization

PubMed Central

Khelashvili, George; Weinstein, Harel; Harries, Daniel

2008-01-01

As charged macromolecules adsorb and diffuse on cell membranes in a large variety of cell signaling processes, they can attract or repel oppositely charged lipids. This results in lateral membrane rearrangement and affects the dynamics of protein function. To address such processes quantitatively we introduce a dynamic mean-field scheme that allows self-consistent calculations of the equilibrium state of membrane-protein complexes after such lateral reorganization of the membrane components, and serves to probe kinetic details of the process. Applicable to membranes with heterogeneous compositions containing several types of lipids, this comprehensive method accounts for mobile salt ions and charged macromolecules in three dimensions, as well as for lateral demixing of charged and net-neutral lipids in the membrane plane. In our model, the mobility of membrane components is governed by the diffusion-like Cahn-Hilliard equation, while the local electrochemical potential is based on nonlinear Poisson-Boltzmann theory. We illustrate the method by applying it to the adsorption of the anionic polypeptide poly-Lysine on negatively charged lipid membranes composed of binary mixtures of neutral and monovalent lipids, or onto ternary mixtures of neutral, monovalent, and multivalent lipids. Consistent with previous calculations and experiments, our results show that at steady-state multivalent lipids (such as PIP2), but not monovalent lipid (such as phosphatidylserine), will segregate near the adsorbing macromolecules. To address the corresponding diffusion of the adsorbing protein in the membrane plane, we couple lipid mobility with the propagation of the adsorbing protein through a dynamic Monte Carlo scheme. We find that due to their higher mobility dictated by the electrochemical potential, multivalent lipids such as PIP2 more quickly segregate near oppositely charged proteins than do monovalent lipids, even though their diffusion constants may be similar. The segregation, in turn, slows protein diffusion, as lipids introduce an effective drag on the motion of the adsorbate. In contrast, monovalent lipids such as phosphatidylserine only weakly segregate, and the diffusions of protein and lipid remain largely uncorrelated. PMID:18065451

Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles

PubMed Central

Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

2015-01-01

The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport. PMID:26131551

Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

PubMed

Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

2015-01-01

The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

PubMed Central

Peccia, Jordan; Marchand, Eric A.; Silverstein, Joann; Hernandez, Mark

2000-01-01

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using 32P radiolabels, probe specificity was characterized by hybridization dissociation temperature (Td) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined Tds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris. PMID:10877807

Nanospectrofluorometry inside single living cell by scanning near-field optical microscopy

NASA Astrophysics Data System (ADS)

Lei, F. H.; Shang, G. Y.; Troyon, M.; Spajer, M.; Morjani, H.; Angiboust, J. F.; Manfait, M.

2001-10-01

Near-field fluorescence spectra with subdiffraction limit spatial resolution have been taken in the proximity of mitochondrial membrane inside breast adenocarcinoma cells (MCF7) treated with the fluorescent dye (JC-1) by using a scanning near-field optical microscope coupled with a confocal laser microspectrofluorometer. The probe-sample distance control is based on a piezoelectric bimorph shear force sensor having a static spring constant k=5 μN/nm and a quality factor Q=40 in a physiological medium of viscosity η=1.0 cp. The sensitivity of the force sensor has been tested by imaging a MCF7 cell surface.

Highly Sensitive Temperature Sensors Based on Fiber-Optic PWM and Capacitance Variation Using Thermochromic Sensing Membrane.

PubMed

Khan, Md Rajibur Rahaman; Kang, Shin-Won

2016-07-09

In this paper, we propose a temperature/thermal sensor that contains a Rhodamine-B sensing membrane. We applied two different sensing methods, namely, fiber-optic pulse width modulation (PWM) and an interdigitated capacitor (IDC)-based temperature sensor to measure the temperature from 5 °C to 100 °C. To the best of our knowledge, the fiber-optic PWM-based temperature sensor is reported for the first time in this study. The proposed fiber-optic PWM temperature sensor has good sensing ability; its sensitivity is ~3.733 mV/°C. The designed temperature-sensing system offers stable sensing responses over a wide dynamic range, good reproducibility properties with a relative standard deviation (RSD) of ~0.021, and the capacity for a linear sensing response with a correlation coefficient of R² ≈ 0.992 over a wide sensing range. In our study, we also developed an IDC temperature sensor that is based on the capacitance variation principle as the IDC sensing element is heated. We compared the performance of the proposed temperature-sensing systems with different fiber-optic temperature sensors (which are based on the fiber-optic wavelength shift method, the long grating fiber-optic Sagnac loop, and probe type fiber-optics) in terms of sensitivity, dynamic range, and linearity. We observed that the proposed sensing systems have better sensing performance than the above-mentioned sensing system.

Actin-based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol.

PubMed

Cheng, Mandy I; Chen, Chen; Engström, Patrik; Portnoy, Daniel A; Mitchell, Gabriel

2018-05-03

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin-based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin-based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time-lapse microscopy using green fluorescent protein-LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin-based motility moved away from LC3-positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin-based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol. © 2018 John Wiley & Sons Ltd.

Effective and efficient detection of pH fluctuations based on ratiometric metallic-ciprofloxacin architectures

NASA Astrophysics Data System (ADS)

Wang, Zhuosen; Gao, Jinwei; Zhang, Kaibo; Mai, Zhihong; Wang, Qianming

2018-07-01

The availability of lanthanide ciprofloxacin complexes and the exploration of efficient new ways to the target species have made fluorescent signals as essential tools for chemical sensing. Both terbium (III) and europium (III) compounds possess easily distinguished, line-like emission bands occurring in the green and red region respectively. Based on the steps of ionizations and the coordination structure changes, the two molecular probes give rise to unique pH-sensitivities at different conditions. The photoluminescence properties of the mixture for the two complexes are demonstrated. At pH from 3 to 6, the Eu(III) emission is found to be less affected and the solution emits blue light in acidic environment (pH = 3). The terbium (III) characteristic luminescence exhibited off-on changes within a narrow pH range (pH = 5-6). Further spectroscopic pH titrations (pH from 6 to 10) are performed and the Eu (III) red emission has been significantly improved. The molecular-based probes have excellent water solubility, negligible cytotoxicity and enough permeability to across cell membrane. Such pH-responsive performance has been carried out for the investigation of intracellular pH measurement and these novel pH indicators were considered to be suitable for detecting bio-medical samples.

A self-assembled fluorescent organic nanoprobe and its application for sulfite detection in food samples and living systems.

PubMed

Gao, Tang; Cao, Xiaozheng; Ge, Peng; Dong, Jie; Yang, Shuqi; Xu, Huan; Wu, Yong; Gao, Feng; Zeng, Wenbin

2017-05-23

Sulfur dioxide (SO 2 ) is a widely distributed air pollutant, and humans can easily be exposed to sulfite by inhaling SO 2 , thus inducing respiratory responses and diseases. Hence, to develop a rapid, sensitive and selective method for detection of sulfites is of great importance. Herein, we designed and synthesized a novel tetraphenyl imidazole compound TIBM with aggregation-induced emission enhancement (AIEE). TIBM can self-assemble into well-organized nanoparticles and is reported as an excellent probe for detection of sulfite with high selectivity and sensitivity. The nanoprobe performed very well for the detection of sulfite with an ultrafast detection time (15 s) and an ultralow detection limit (7.4 nM), which is superior to most of the reported probes. Moreover, the nanoprobe was successfully used to detect sulfite in food samples with a favorable accuracy. In addition, we developed paper-based devices for point-of-care detection of sulfite with naked eyes. Furthermore, due to its high water solubility, cell membrane permeability and good biocompatibility, the nanoproboe was further applied to detect sulfite in living systems. This study may offer some helpful insights for designing other AIE-based fluorescent nanosensors for various analytes.

Light-Addressable Measurement of in Vivo Tissue Oxygenation in an Unanesthetized Zebrafish Embryo via Phase-Based Phosphorescence Lifetime Detection

PubMed Central

Huang, Shih-Hao; Yu, Chu-Hung; Chien, Yi-Lung

2015-01-01

We have developed a digital light modulation system that utilizes a modified commercial projector equipped with a laser diode as a light source for quantitative measurements of in vivo tissue oxygenation in an unanesthetized zebrafish embryo via phase-based phosphorescence lifetime detection. The oxygen-sensitive phosphorescent probe (Oxyphor G4) was first inoculated into the bloodstream of 48 h post-fertilization (48 hpf) zebrafish embryos via the circulation valley to rapidly disperse probes throughout the embryo. The unanesthetized zebrafish embryo was introduced into the microfluidic device and immobilized on its lateral side by using a pneumatically actuated membrane. By controlling the illumination pattern on the digital micromirror device in the projector, the modulated excitation light can be spatially projected to illuminate arbitrarily-shaped regions of tissue of interest for in vivo oxygen measurements. We have successfully measured in vivo oxygen changes in the cardiac region and cardinal vein of a 48 hpf zebrafish embryo that experience hypoxia and subsequent normoxic conditions. Our proposed platform provides the potential for the real-time investigation of oxygen distribution in tissue microvasculature that relates to physiological stimulation and diseases in a developing organism. PMID:25856326

In vivo study for the discrimination of cancerous and normal skin using fibre probe-based Raman spectroscopy.

PubMed

Schleusener, Johannes; Gluszczynska, Patrycja; Reble, Carina; Gersonde, Ingo; Helfmann, Jürgen; Fluhr, Joachim W; Lademann, Jürgen; Röwert-Huber, Joachim; Patzelt, Alexa; Meinke, Martina C

2015-10-01

Raman spectroscopy has proved its capability as an objective, non-invasive tool for the detection of various melanoma and non-melanoma skin cancers (NMSC) in a number of studies. Most publications are based on a Raman microspectroscopic ex vivo approach. In this in vivo clinical evaluation, we apply Raman spectroscopy using a fibre-coupled probe that allows access to a multitude of affected body sites. The probe design is optimized for epithelial sensitivity, whereby a large part of the detected signal originates from within the epidermal layer's depth down to the basal membrane where early stages of skin cancer develop. Data analysis was performed on measurements of 104 subjects scheduled for excision of lesions suspected of being malignant melanoma (MM) (n = 36), basal cell carcinoma (BCC) (n = 39) and squamous cell carcinoma (SCC) (n = 29). NMSC were discriminated from normal skin with a balanced accuracy of 73% (BCC) and 85% (SCC) using partial least squares discriminant analysis (PLS-DA). Discriminating MM and pigmented nevi (PN) resulted in a balanced accuracy of 91%. These results lie within the range of comparable in vivo studies and the accuracies achieved by trained dermatologists using dermoscopy. Discrimination proved to be unsuccessful between cancerous lesions and suspicious lesions that had been histopathologically verified as benign by dermoscopy. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Interaction of amphotericin B and its low toxic derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method.

PubMed

Szlinder-Richert, Joanna; Cybulska, Barbara; Grzybowska, Jolanta; Bolard, Jacques; Borowski, Edward

2004-04-01

Amphotericin B (AMB) derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

PubMed

Murota, Yoshitaka; Tabu, Kouichi; Taga, Tetsuya

2016-11-04

Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable. Inhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Electrochemical reverse engineering: A systems-level tool to probe the redox-based molecular communication of biology.

PubMed

Li, Jinyang; Liu, Yi; Kim, Eunkyoung; March, John C; Bentley, William E; Payne, Gregory F

2017-04-01

The intestine is the site of digestion and forms a critical interface between the host and the outside world. This interface is composed of host epithelium and a complex microbiota which is "connected" through an extensive web of chemical and biological interactions that determine the balance between health and disease for the host. This biology and the associated chemical dialogues occur within a context of a steep oxygen gradient that provides the driving force for a variety of reduction and oxidation (redox) reactions. While some redox couples (e.g., catecholics) can spontaneously exchange electrons, many others are kinetically "insulated" (e.g., biothiols) allowing the biology to set and control their redox states far from equilibrium. It is well known that within cells, such non-equilibrated redox couples are poised to transfer electrons to perform reactions essential to immune defense (e.g., transfer from NADH to O 2 for reactive oxygen species, ROS, generation) and protection from such oxidative stresses (e.g., glutathione-based reduction of ROS). More recently, it has been recognized that some of these redox-active species (e.g., H 2 O 2 ) cross membranes and diffuse into the extracellular environment including lumen to transmit redox information that is received by atomically-specific receptors (e.g., cysteine-based sulfur switches) that regulate biological functions. Thus, redox has emerged as an important modality in the chemical signaling that occurs in the intestine and there have been emerging efforts to develop the experimental tools needed to probe this modality. We suggest that electrochemistry provides a unique tool to experimentally probe redox interactions at a systems level. Importantly, electrochemistry offers the potential to enlist the extensive theories established in signal processing in an effort to "reverse engineer" the molecular communication occurring in this complex biological system. Here, we review our efforts to develop this electrochemical tool for in vitro redox-probing. Copyright © 2017 Elsevier Inc. All rights reserved.

In vivo imaging of membrane type-1 matrix metalloproteinase with a novel activatable near-infrared fluorescence probe.

PubMed

Shimizu, Yoichi; Temma, Takashi; Hara, Isao; Makino, Akira; Kondo, Naoya; Ozeki, Ei-Ichi; Ono, Masahiro; Saji, Hideo

2014-08-01

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease activating MMP-2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1-MMP would be useful for tumor diagnosis, we developed a novel near-infrared (NIR) fluorescence probe that can be activated following interaction with MT1-MMP in vivo. MT1-hIC7L is an activatable fluorescence probe comprised of anti-MT1-MMP monoclonal antibodies conjugated to self-assembling polymer micelles that encapsulate NIR dyes (IC7-1, λem : 858 nm) at concentrations sufficient to cause fluorescence self-quenching. In aqueous buffer, MT1-hIC7L fluorescence was suppressed to background levels and increased approximately 35.5-fold in the presence of detergent. Cellular uptake experiments revealed that in MT1-MMP positive C6 glioma cells, MT1-hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti-MT1-MMP monoclonal antibody. In MT1-MMP negative MCF-7 breast adenocarcinoma cells, both MT1-hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1-hIC7L was suppressed by pre-treatment with an MT1-MMP endocytosis inhibitor (P < 0.05). In vivo optical imaging using probes intravenously administered to tumor-bearing mice showed that MT1-hIC7L specifically visualized C6 tumors (tumor-to-background ratios: 3.8 ± 0.3 [MT1-hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P < 0.05), while the probes showed similarly low fluorescence in MCF-7 tumors. Together, these results show that MT1-hIC7L would be a potential activatable NIR probe for specifically detecting MT1-MMP-expressing tumors. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Coordinated Mechanosensitivity of Membrane Rafts and Focal Adhesions

PubMed Central

Fuentes, Daniela E.; Butler, Peter J.

2013-01-01

Endothelial cells sense mechanical forces of blood flow through mechanisms that involve focal adhesions (FAs). The mechanosensitive pathways that originate from FA-associated integrin activation may involve membrane rafts, small cholesterol- and sphigolipid-rich domains that are either immobilized, by virtue of their attachment to the cytoskeleton, or highly mobile in the plane of the plasma membrane. In this study, we fluorescently labeled non-mobile and mobile populations of GM1, a ganglioside associated with lipid rafts, and transfected cells with the red fluorescent protein-(RFP-) talin, an indicator of integrin activation at FAs, in order to determine the kinetics and sequential order of raft and talin mechanosensitivity. Cells were imaged under confocal microscopy during mechanical manipulation of a FA induced by a fibronectin (FN)-functionalized nanoelectrode with feedback control of position. First, FA deformation led to long range deformation of immobile rafts followed by active recoil of a subpopulation of displaced rafts. Second, initial adhesion between the FN-probe and the cell induced rapid accumulation of GM1 at the probe site with a time constant of 1.7 s. Talin accumulated approximately 20 s later with a time constant of 0.6 s. Third, a 1 μm deformation of the FA lead to immediate (0.3 s) increase in GM1 fluorescence and a later (6 s) increase in talin. Fourth, long term deformation of FAs led to continual GM1 accumulation at the probe site that was reversed upon removal of the deformation. These results demonstrate that rafts are directly mechanosensitive and that raft mobility may enable the earliest events related to FA mechanosensing and reinforcement upon force application. PMID:23487555

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

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

Price, E.M.; Freisheim, J.H.

1987-07-28

A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake,more » with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.« less

Effect of vesicle size on the prodan fluorescence in diheptadecanoylphosphatidylcholine bilayer membrane under atmospheric and high pressures.

PubMed

Goto, Masaki; Sawaguchi, Hiroshi; Tamai, Nobutake; Matsuki, Hitoshi; Kaneshina, Shoji

2010-08-17

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they become a useful membrane probe, detecting important membrane properties such as the packing stress.

A fiber-optic sensor for accurately monitoring biofilm growth in a hydrogen production photobioreactor.

PubMed

Zhong, Nianbing; Liao, Qiang; Zhu, Xun; Chen, Rong

2014-04-15

A new simple fiber-optic evanescent wave sensor was created to accurately monitor the growth and hydrogen production performance of biofilms. The proposed sensor consists of two probes (i.e., a sensor and reference probe), using the etched fibers with an appropriate surface roughness to improve its sensitivity. The sensor probe measures the biofilm growth and change of liquid-phase concentration inside the biofilm. The reference probe is coated with a hydrophilic polytetrafluoroethylene membrane to separate the liquids from photosynthetic bacteria Rhodopseudomonas palustris CQK 01 and to measure the liquid concentration. We also developed a model to demonstrate the accuracy of the measurement. The biofilm measurement was calibrated using an Olympus microscope. A linear relationship was obtained for the biofilm thickness range from 0 to 120 μm with a synthetic medium under continuous supply to the bioreactor. The highest level of hydrogen production rate occurred at a thickness of 115 μm.

Microdialysis of glucose in subcutaneous adipose tissue up to 3 weeks in healthy volunteers.

PubMed

Wientjes, K J; Vonk, P; Vonk-van Klei, Y; Schoonen, A J; Kossen, N W

1998-09-01

To measure possible changes in dialysate glucose concentrations over time, to validate the diffusional model for glucose transport from tissue to the probe, and to evaluate the actual glucose concentration in adipose tissue. Glucose concentrations in the subcutaneous adipose tissue of five healthy subjects (age 25 +/- 2.7 years, BMI 23.2 +/- 2.3 kg/m2 [mean +/- SD]) were measured by the microdialysis technique and compared with blood glucose. We applied microdialysis probes with hollow fibers of various membrane length (10-35 mm), used eight perfusion flow rates (0.5-20 microl/min), and perfused four glucose solutions (0.0, 2.8, 8.3, 11.1 mmol/l). After implantation, a substantial decrease in glucose recovery to the lowest value of 26 +/- 10% of the final plateau value was noted during the first few hours (n = 4). Recovery increased and stabilized after 5-9 days at 84.0 +/- 7.4% of capillary blood glucose when a flow rate of 0.5 microl/min was applied. According to the zero net-flux method, the glucose concentration in equilibrium, Cequi, with the surrounding tissue can be obtained. This concentration also decreases; however, 1 h after recovery, Cequi increases again over 1 or 2 days to a stable value that is not significantly different from the measured capillary blood glucose (P < 0.05). Using various perfusion flow rates and probes (membrane length 10-35 mm), it is shown that diffusion is the rate-limiting process for glucose transport through tissue. Insertion of the microdialysis probes causes damage to the adipose cells and the vascular bed around the probe. Glucose recovery decreases because of a lower blood supply. In 5-9 days, glucose recovery increases; apparently, this time is needed to repair the microstructure of tissue around the probe. After stabilization of the recovery, no loss of probe permeability, which is due to biocompatibility problems, was seen. The change during the 2 days in equilibrium concentration is probably caused by an inflammation reaction that consumes glucose around the probe. The individual increase in recovery during the 1st days after probe insertion until a stable plateau value is reached (flow rate >0 microl/min) is complicated for short-term clinical glucose measurements in adipose tissue. After stabilization, the mean equilibrium concentration of all subjects was equal to the mean capillary blood glucose concentration. Therefore, we conclude that capillary blood glucose concentration probably is the driving force for diffusion through the capillary wall into the probe and is not some interstitial concentration.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

PubMed Central

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar

2015-01-01

ABSTRACT Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress. PMID:26330516

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

PubMed Central

Taraska, Justin W.; Puljung, Michael C.; Zagotta, William N.

2009-01-01

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor–acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements. PMID:19805285

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones.

PubMed

Mark, Christina; Zór, Kinga; Heiskanen, Arto; Dufva, Martin; Emnéus, Jenny; Finnie, Christine

2016-12-15

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential Effect of Plant Lipids on Membrane Organization

PubMed Central

Grosjean, Kevin; Mongrand, Sébastien; Beney, Laurent; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2015-01-01

The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains. PMID:25575593