Addendum: Development of a preprototype times wastewater recovery subsystem

NASA Technical Reports Server (NTRS)

Dehner, G. F.

1984-01-01

The results of the second generation operational improvements and the TIMES (Thermoelectric Integrated Membrane Evaporation Subsystem) 2 study are covered. Areas covered in the second generation operational improvements are improved temperature control, water quality improvements, subsytem operational improvements, solid handling improvements, wastewater pretreatment optimization, and membrane rejuvenation concepts. The task for the TIMES 2 study are thermoelectric regenerator improvement, recycle loop pH operational criteria, recycle loop component optimization, and hollow fiber membrane evaporator improvement. Results are presented and conclusions are drawn from both studies.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

PubMed

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-05-06

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

PubMed Central

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-01-01

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines. PMID:27150638

Pervaporation of model acetone-butanol-ethanol fermentation product solutions using polytetrafluoroethylene membranes

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

Vrana, D.L.; Meagher, M.M.; Hutkins, R.W.

1993-10-01

A pervaporation apparatus was designed and tested in an effort to develop an integrated fermentation and product recovery process for acetone-butanol-ethanol(ABE) fermentation. A crossflow membrane module able to accommodate flat sheet hydrophobic membranes was used for the experiments. Permeate vapors were collected under vacuum and condensed in a dry ice/ethanol cold trap. The apparatus containing polytetrafluoroethylene membranes was tested using butanol-water and model solutions of ABE products. Parameters such as product concentration, component effect, temperature, and permeate side pressure were examined. 25 refs., 3 figs., 5 tabs.

Membrane oxygenator heat exchanger failure detected by unique blood gas findings.

PubMed

Hawkins, Justin L

2014-03-01

Failure of components integrated into the cardiopulmonary bypass circuit, although rare, can bring about catastrophic results. One of these components is the heat exchanger of the membrane oxygenator. In this compartment, unsterile water from the heater cooler device is separated from the sterile blood by stainless steel, aluminum, or by polyurethane. These areas are glued or welded to keep the two compartments separate, maintaining sterility of the blood. Although quality control testing is performed by the manufacturer at the factory level, transport presents the real possibility for damage. Because of this, each manufacturer has included in the instructions for use a testing procedure for testing the integrity of the heat exchanger component. Water is circulated through the heat exchanger before priming and a visible check is made of the oxygenator bundle to check for leaks. If none are apparent, then priming of the oxygenator is performed. In this particular case, this procedure was not useful in detecting communication between the water and blood chambers of the oxygenator.

Integral design method for simple and small Mars lander system using membrane aeroshell

NASA Astrophysics Data System (ADS)

Sakagami, Ryo; Takahashi, Ryohei; Wachi, Akifumi; Koshiro, Yuki; Maezawa, Hiroyuki; Kasai, Yasko; Nakasuka, Shinichi

2018-03-01

To execute Mars surface exploration missions, spacecraft need to overcome the difficulties of the Mars entry, descent, and landing (EDL) sequences. Previous landing missions overcame these challenges with complicated systems that could only be executed by organizations with mature technology and abundant financial resources. In this paper, we propose a novel integral design methodology for a small, simple Mars lander that is achievable even by organizations with limited technology and resources such as universities or emerging countries. We aim to design a lander (including its interplanetary cruise stage) whose size and mass are under 1 m3 and 150 kg, respectively. We adopted only two components for Mars EDL process: a "membrane aeroshell" for the Mars atmospheric entry and descent sequence and one additional mechanism for the landing sequence. The landing mechanism was selected from the following three candidates: (1) solid thrusters, (2) aluminum foam, and (3) a vented airbag. We present a reasonable design process, visualize dependencies among parameters, summarize sizing methods for each component, and propose the way to integrate these components into one system. To demonstrate the effectiveness, we applied this methodology to the actual Mars EDL mission led by the National Institute of Information and Communications Technology (NICT) and the University of Tokyo. As a result, an 80 kg class Mars lander with a 1.75 m radius membrane aeroshell and a vented airbag was designed, and the maximum landing shock that the lander will receive was 115 G.

Effect of a Vietnamese Cinnamomum cassia essential oil and its major component trans-cinnamaldehyde on the cell viability, membrane integrity, membrane fluidity, and proton motive force of Listeria innocua.

PubMed

Trinh, Nga-Thi-Thanh; Dumas, Emilie; Thanh, Mai Le; Degraeve, Pascal; Ben Amara, Chedia; Gharsallaoui, Adem; Oulahal, Nadia

2015-04-01

The antibacterial mechanism of a Cinnamomum cassia essential oil from Vietnam and of its main component (trans-cinnamaldehyde, 90% (m/m) of C. cassia essential oil) against a Listeria innocua strain was investigated to estimate their potential for food preservation. In the presence of C. cassia essential oil or trans-cinnamaldehyde at their minimal bactericidal concentration (2700 μg·mL(-1)), L. innocua cells fluoresced green after staining with Syto9® and propidium iodide, as observed by epifluorescence microscopy, suggesting that the perturbation of membrane did not cause large pore formation and cell lysis but may have introduced the presence of viable but nonculturable bacteria. Moreover, the fluidity, potential, and intracellular pH of the cytoplasmic membrane were perturbed in the presence of the essential oil or trans-cinnamaldehyde. However, these membrane perturbations were less severe in the presence of trans-cinnamaldehyde than in the presence of multicomponent C. cassia essential oil. This indicates that in addition to trans-cinnamaldehyde, other minor C. cassia essential oil components play a major role in its antibacterial activity against L. innocua cells.

Membrane preparation and solubilization.

PubMed

Roy, Ankita

2015-01-01

Membrane proteins play an essential role in several biological processes like ion transport, signal transduction, and electron transfer to name a few. For structural and functional studies of integral membrane proteins, it is critically important to isolate proteins from the membrane using biological detergents. Detergents disrupt the native lipid components of the native membrane and encase the membrane protein in an unnatural environment in aqueous solution. However, a particular membrane protein is best solubilized in a specific detergent; therefore, screening for the optimal detergent is essential. Apart from keeping the membrane protein monodispered in solution, the detergent has to be compatible with downstream processes to isolate and characterize a membrane protein. Over the past several years, a number of membrane proteins have been successfully isolated for structural and functional studies that allowed an outline of general strategies for isolating a novel membrane protein of interest. © 2015 Elsevier Inc. All rights reserved.

Type IV Collagens and Basement Membrane Diseases: Cell Biology and Pathogenic Mechanisms.

PubMed

Mao, Mao; Alavi, Marcel V; Labelle-Dumais, Cassandre; Gould, Douglas B

2015-01-01

Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Stabilization of apoptotic cells: generation of zombie cells.

PubMed

Oropesa-Ávila, M; Andrade-Talavera, Y; Garrido-Maraver, J; Cordero, M D; de la Mata, M; Cotán, D; Paz, M V; Pavón, A D; Alcocer-Gómez, E; de Lavera, I; Lema, R; Zaderenko, A P; Rodríguez-Moreno, A; Sánchez-Alcázar, J A

2014-08-14

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn(2+) (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.

Stabilization of apoptotic cells: generation of zombie cells

PubMed Central

Oropesa-Ávila, M; Andrade-Talavera, Y; Garrido-Maraver, J; Cordero, M D; de la Mata, M; Cotán, D; Paz, M V; Pavón, A D; Alcocer-Gómez, E; de Lavera, I; Lema, R; Zaderenko, A P; Rodríguez-Moreno, A; Sánchez-Alcázar, J A

2014-01-01

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy. PMID:25118929

Identification of Two Novel Endoplasmic Reticulum Body-Specific Integral Membrane Proteins1[W][OA

PubMed Central

Yamada, Kenji; Nagano, Atsushi J.; Nishina, Momoko; Hara-Nishimura, Ikuko; Nishimura, Mikio

2013-01-01

The endoplasmic reticulum (ER) body, a large compartment specific to the Brassicales, accumulates β-glucosidase and possibly plays a role in the defense against pathogens and herbivores. Although the ER body is a subdomain of the ER, it is unclear whether any ER body-specific membrane protein exists. In this study, we identified two integral membrane proteins of the ER body in Arabidopsis (Arabidopsis thaliana) and termed them MEMBRANE PROTEIN OF ENDOPLASMIC RETICULUM BODY1 (MEB1) and MEB2. In Arabidopsis, a basic helix-loop-helix transcription factor, NAI1, and an ER body component, NAI2, regulate ER body formation. The expression profiles of MEB1 and MEB2 are similar to those of NAI1, NAI2, and ER body β-glucosidase PYK10 in Arabidopsis. The expression of MEB1 and MEB2 was reduced in the nai1 mutant, indicating that NAI1 regulates the expression of MEB1 and MEB2 genes. MEB1 and MEB2 proteins localize to the ER body membrane but not to the ER network, suggesting that these proteins are specifically recruited to the ER body membrane. MEB1 and MEB2 physically interacted with ER body component NAI2, and they were diffused throughout the ER network in the nai2 mutant, which has no ER body. Heterologous expression of MEB1 and MEB2 in yeast (Saccharomyces cerevisiae) suppresses iron and manganese toxicity, suggesting that MEB1 and MEB2 are metal transporters. These results indicate that the membrane of ER bodies has specific membrane proteins and suggest that the ER body is involved in defense against metal stress as well as pathogens and herbivores. PMID:23166355

Intrinsic reaction-cycle time scale of Na+,K+-ATPase manifests itself in the lipid–protein interactions of nonequilibrium membranes

PubMed Central

Bouvrais, Hélène; Cornelius, Flemming; Ipsen, John H.; Mouritsen, Ole G.

2012-01-01

Interaction between integral membrane proteins and the lipid-bilayer component of biological membranes is expected to mutually influence the proteins and the membrane. We present quantitative evidence of a manifestation of the lipid–protein interactions in liposomal membranes, reconstituted with actively pumping Na+,K+-ATPase, in terms of nonequilibrium shape fluctuations that contain a relaxation time, τ, which is robust and independent of the specific fluctuation modes of the membrane. In the case of pumping Na+-ions, analysis of the flicker-noise temporal correlation spectrum of the liposomes leads to τ ≃ 0.5 s, comparing favorably with an intrinsic reaction-cycle time of about 0.4 s from enzymology. PMID:23093677

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

PubMed

Tan, Zaigao; Yoon, Jong Moon; Nielsen, David R; Shanks, Jacqueline V; Jarboe, Laura R

2016-05-01

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

PubMed Central

Shotton, D.; Thompson, K.; Wofsy, L.; Branton, D.

1978-01-01

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others. PMID:10605454

Association of Membrane Rafts and Postsynaptic Density: Proteomics, Biochemical, and Ultrastructural Analyses

PubMed Central

Suzuki, Tatsuo; Zhang, Jingping; Miyazawa, Shoko; Liu, Qian; Farzan, Michael R.; Yao, Wei-Dong

2011-01-01

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. However, their molecular identities remain elusive. Further, how they interact with the well-established signaling specialization, the postsynaptic density (PSD), is poorly understood. We previously detected a number of conventional PSD proteins in detergent-resistant membranes (DRMs). Here, we have performed LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) analyses on postsynaptic membrane rafts and PSDs. Our comparative analysis identified an extensive overlap of protein components in the two structures. This overlapping could be explained, at least partly, by a physical association of the two structures. Meanwhile, a significant number of proteins displayed biased distributions to either rafts or PSDs, suggesting distinct roles for the two postsynaptic specializations. Using biochemical and electron microscopic methods, we directly detected membrane raft-PSD complexes. In vitro reconstitution experiments indicated that the formation of raft-PSD complexes was not due to the artificial reconstruction of once-solubilized membrane components and PSD structures, supporting that these complexes occurred in vivo. Taking together, our results provide evidence that postsynaptic membrane rafts and PSDs may be physically associated. Such association could be important in postsynaptic signal integration, synaptic function, and maintenance. PMID:21797867

Possible role for fundus autofluorescence as a predictive factor for visual acuity recovery after epiretinal membrane surgery.

PubMed

Brito, Pedro N; Gomes, Nuno L; Vieira, Marco P; Faria, Pedro A; Fernandes, Augusto V; Rocha-Sousa, Amândio; Falcão-Reis, Fernando

2014-02-01

To study the potential association between fundus autofluorescence, spectral-domain optical coherence tomography, and visual acuity in patients undergoing surgery because of epiretinal membranes. Prospective, interventional case series including 26 patients submitted to vitrectomy because of symptomatic epiretinal membranes. Preoperative evaluation consisted of a complete ophthalmologic examination, autofluorescence, and spectral-domain optical coherence tomography. Studied variables included foveal autofluorescence (fov.AF), photoreceptor inner segment/outer segment (IS/OS) junction line integrity, external limiting membrane integrity, central foveal thickness, and foveal morphology. All examinations were repeated at the first, third, and sixth postoperative months. The main outcome measures were logarithm of minimal angle resolution visual acuity, fov.AF integrity, and IS/OS integrity. All cases showing a continuous IS/OS line had an intact fov.AF, whereas patients with IS/OS disruption could have either an increased area of foveal hypoautofluorescence or an intact fov.AF, with the latter being associated with IS/OS integrity recovery in follow-up spectral-domain optical coherence tomography imaging. The only preoperative variables presenting a significant correlation with final visual acuity were baseline visual acuity (P = 0.047) and fov.AF grade (P = 0.023). Recovery of IS/OS line integrity after surgery, in patients with preoperative IS/OS disruption and normal fov.AF, can be explained by the presence of a functional retinal pigment epithelium-photoreceptor complex, supporting normal photoreceptor activity. Autofluorescence imaging provides a functional component to the study of epiretinal membranes, complementing the structural information obtained with optical coherence tomography.

The Investigation and Development of Low Cost Hardware Components for Proton-Exchange Membrane Fuel Cells - Final Report

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

George A. Marchetti

1999-12-15

Proton exchange membrane (PEM) fuel cell components, which would have a low-cost structure in mass production, were fabricated and tested. A fuel cell electrode structure, comprising a thin layer of graphite (50 microns) and a front-loaded platinum catalyst layer (600 angstroms), was shown to produce significant power densities. In addition, a PEM bipolar plate, comprising flexible graphite, carbon cloth flow-fields and an integrated polymer gasket, was fabricated. Power densities of a two-cell unit using this inexpensive bipolar plate architecture were shown to be comparable to state-of-the-art bipolar plates.

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

PubMed Central

Summer, Elizabeth J.; Cline, Kenneth

1999-01-01

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways. PMID:9952453

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.

PubMed

Wenz, Lena-Sophie; Opaliński, Lukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

2014-06-01

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. © 2014 The Authors.

Involvement of vesicle coat material in casein secretion and surface regeneration

PubMed Central

1976-01-01

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces. PMID:1254641

METHODS STUDIES FOR THE NATIONAL CHILDREN'S STUDY: SEMIPERMEABLE MEMBRANE DEVICE (SPMD)

EPA Science Inventory

Accurate exposure classification tools are required to link exposure with health effects in epidemiological studies. Although long-term integrated exposure measurements are a critical component of exposure assessment, the ability to include these measurements into epidemiologic...

Characterization of membrane association of Rinderpest virus matrix protein

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

Subhashri, R.; Shaila, M.S.

2007-04-20

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M proteinmore » gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.« less

A high-performance polydimethylsiloxane electrospun membrane for cell culture in lab-on-a-chip.

PubMed

Moghadas, Hajar; Saidi, Mohammad Said; Kashaninejad, Navid; Nguyen, Nam-Trung

2018-03-01

Thin porous membranes are important components in a microfluidic device, serving as separators, filters, and scaffolds for cell culture. However, the fabrication and the integration of these membranes possess many challenges, which restrict their widespread applications. This paper reports a facile technique to fabricate robust membrane-embedded microfluidic devices. We integrated an electrospun membrane into a polydimethylsiloxane (PDMS) device using the simple plasma-activated bonding technique. To increase the flexibility of the membrane and to address the leakage problem, the electrospun membrane was fabricated with the highest weight ratio of PDMS to polymethylmethacrylate (i.e., 6:1 w/w). The membrane-integrated microfluidic device could withstand a flow rate of up to 50  μ l/min. As a proof of concept, we demonstrated that such a compartmentalized microfluidic platform could be successfully used for cell culture with the capability of providing a more realistic in vivo -like condition. Human lung cancer epithelial cells (A549) were seeded on the membrane from the top microchannel, while the continuous flow of the culture medium through the bottom microchannel provided a shear-free cell culture condition. The tortuous micro-/nanofibers of the membrane immobilized the cells within the hydrophobic micropores and with no need of extracellular matrix for cell adhesion and cell growth. The hydrophobic surface conditions of the membrane were suitable for anchorage-independent cell types. To further extend the application of the device, we qualitatively showed that rinsing the membrane with ethanol prior to cell seeding could temporarily render the membrane hydrophilic and the platform could also be used for anchorage-dependent cells. Due to the three-dimensional (3D) topography of the membranes, three different configurations were observed, including individual single cells, monolayer cells, and 3D cell clusters. This cost-effective and robust compartmentalized microfluidic device may open up new avenues in translational medicine and pharmacodynamics research.

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis.

PubMed

Sánchez-Alcázar, José A; Rodríguez-Hernández, Angeles; Cordero, Mario D; Fernández-Ayala, Daniel J M; Brea-Calvo, Gloria; Garcia, Katherina; Navas, Plácido

2007-07-01

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional gamma-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon". Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to d-Limonene Show Changes to the Cell Wall but Not to the Plasma Membrane

PubMed Central

Brennan, Timothy C. R.; Nielsen, Lars K.

2013-01-01

Monoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae, has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d-limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes (ROM1, RLM1, PIR3, CTT1, YGP1, MLP1, PST1, and CWP1) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis. PMID:23542628

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

PubMed Central

Kirchhausen, Tom; Owen, David; Harrison, Stephen C.

2014-01-01

Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation. PMID:24789820

The Fluid-Mosaic Model of Membrane Structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years.

PubMed

Nicolson, Garth L

2014-06-01

In 1972 the Fluid-Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720-731]. After over 40years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid-lipid, protein-protein and lipid-protein interactions as well as cell-matrix, cell-cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along with membrane-associated cytoskeletal and extracellular structures maintain the long-range, non-random mosaic macro-organization of membranes, while smaller membrane nano- and submicro-sized domains, such as lipid rafts and protein complexes, are important in maintaining specialized membrane structures that are in cooperative dynamic flux in a crowded membrane plane. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. © 2013.

Refined views of multi-protein complexes in the erythrocyte membrane

PubMed Central

Mankelow, TJ; Satchwell, TJ; Burton, NM

2015-01-01

The erythrocyte membrane has been extensively studied, both as a model membrane system and to investigate its role in gas exchange and transport. Much is now known about the protein components of the membrane, how they are organised into large multi-protein complexes and how they interact with each other within these complexes. Many links between the membrane and the cytoskeleton have also been delineated and have been demonstrated to be crucial for maintaining the deformability and integrity of the erythrocyte. In this study we have refined previous, highly speculative molecular models of these complexes by including the available data pertaining to known protein-protein interactions. While the refined models remain highly speculative, they provide an evolving framework for visualisation of these important cellular structures at the atomic level. PMID:22465511

Insights into the structure and function of membrane-integrated processive glycosyltransferases

DOE PAGES

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; ...

2015-09-02

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

Insights into the structure and function of membrane-integrated processive glycosyltransferases

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

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

Composition-dependent Membrane Disruption by the Proapoptotic Protein PB1F2 from HK97 Influenza A Virus.

PubMed

Wang, Yujuan; Yang, Jing; Wang, Jiarong; Zhu, Lei; Wang, Junfeng

2018-06-22

PB1F2 is a proapoptotic protein encoded by an alternative reading frame in the influenza A virus. Its accumulation accelerates mitochondrial fragmentation by decreasing the mitochondrial membrane potential following translocation into the mitochondrial inner membrane space, but the mechanistic underpinnings remain unclear. Herein, the PB1F2 from HK97 was expressed and purified in soluble form. The interaction between PB1F2 and the mitochondrial membrane were investigated using three membrane mimics, liposomes, bicelles and nanodiscs. We show that the interactions between PB1F2 and membrane mimics depend on lipid type and are time- and dose-dependent. The primary membrane target of PB1F2 is phosphatidylcholine, the lipid that forms the major component of mitochondrial inner membranes. PB1F2 disrupts the integrity of lipid membranes by forming micelle-like PB1F2-lipid assemblies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

NASA Astrophysics Data System (ADS)

Kuzmenko, Anton; Tankov, Stoyan; English, Brian P.; Tarassov, Ivan; Tenson, Tanel; Kamenski, Piotr; Elf, Johan; Hauryliuk, Vasili

2011-12-01

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

A Prototype Actuator Concept for Membrane Boundary Vibration Control

NASA Technical Reports Server (NTRS)

Solter, Micah J.

2005-01-01

In conjunction with the research in ultra-lightweight deployable spacecraft and membrane structures is an underlying need for shape and vibration control. For thin film membrane structures, fundamental modes of vibration for the membrane can be excited through station keeping, attitude adjustments, orbital maneuvers, or contact with space junk or micrometeorites. In order to maintain structural integrity as well as surface shape contour, which may be essential for inflatable antennas, reflective surfaces, or solar sails; vibration damping is a necessary component. This paper discusses development of an actuator attached at the membrane boundary, containing two types of piezoelectric elements, which can be used to perform active control of vibration from the boundary of a membrane. The actuator is designed to control the membrane out-of-plane displacement and in-plane tension by varying the boundary conditions. Results from an initial experimental evaluation of the concept are presented with bench tests of the actuator alone, and with the actuator connected to a large membrane.

Aspects of nuclear envelope dynamics in mitotic cells.

PubMed

Burke, Brian; Shanahan, Catherine; Salina, Davide; Crisp, Melissa

2005-01-01

Major features of the nuclear envelope (NE) are a pair of inner and outer nuclear membranes (INM, ONM) spanned by nuclear pore complexes. While the composition of the ONM resembles that of the endoplasmic reticulum, the INM contains a unique spectrum of proteins. Localization of INM proteins involves a mechanism of selective retention whereby integral proteins are immobilized and concentrated by virtue of interactions with nuclear components. In the case of emerin, INM localization involves interaction with A-type lamins. Interactions between membrane proteins may also play a significant role in INM localization. This conclusion stems from studies on nesprins, a family of membrane proteins that feature a large cytoplasmic domain, a single C-terminal membrane-spanning domain and a small lumenal domain. The nesprin membrane anchor and lumenal (KASH) domains are related to the Drosophila Klarsicht protein. Evidence is emerging that this KASH region interacts with other NE proteins and may influence their distributions. Overexpression of GFP-KASH causes loss of emerin and LAP2 from the NE. This is not due to global reorganization of the NE since LAP1 as well as lamins and NPCs remain unaffected. Our results suggest that interactions between NE membrane components are far more extensive and complex than current models suggest.

Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

PubMed

Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

2014-01-01

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria.

PubMed

Wrobel, Lidia; Trojanowska, Agata; Sztolsztener, Malgorzata E; Chacinska, Agnieszka

2013-03-01

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.

Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

PubMed

Discher, D E; Mohandas, N

1996-10-01

Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton.

Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

PubMed

Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

2016-10-07

Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

Transcriptomic insights into citrus segment membrane's cell wall components relating to fruit sensory texture.

PubMed

Wang, Xun; Lin, Lijin; Tang, Yi; Xia, Hui; Zhang, Xiancong; Yue, Maolan; Qiu, Xia; Xu, Ke; Wang, Zhihui

2018-04-23

During fresh fruit consumption, sensory texture is one factor that affects the organoleptic qualities. Chemical components of plant cell walls, including pectin, cellulose, hemicellulose and lignin, play central roles in determining the textural qualities. To explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture, we performed mRNA-seq analyses of the segment membranes of two citrus cultivars, Shiranui and Kiyomi, with different organoleptic textures. Segment membranes were sampled at two developmental stages of citrus fruit, the beginning and end of the expansion period. More than 3000 differentially expressed genes were identified. The gene ontology analysis revealed that more categories were significantly enriched in 'Shiranui' than in 'Kiyomi' at both developmental stages. In total, 108 significantly enriched pathways were obtained, with most belonging to metabolism. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis of the segment membranes from mature fruit revealed that the pectin, cellulose and lignin contents, and the segment membrane's weight (% of segment) were greater in 'Kiyomi'. Organoleptic quality of citrus is easily overlooked. It is mainly determined by sensory texture perceived in citrus segment membrane properties. We performed mRNA-seq analyses of citrus segment membranes to explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture. Transcriptomic data showed high repeatability between two independent biological replicates. The expression levels of genes involved in cell wall structure metabolism, including pectin, cellulose, hemicellulose and lignin, were investigated. Meanwhile, chemical component contents of the segment membranes from mature fruit were analyzed. This study provided detailed transcriptional regulatory profiles of different organoleptic citrus qualities and integrated insights into the mechanisms affecting citrus' sensory texture.

Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

PubMed

Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Zhendre, Vanessa; Henry, Sarah; Thévenot, Julie; Dufourc, Erick J; Alves, Isabel D; Peruch, Frédéric

2014-01-01

The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles. © 2013.

A link between mitotic entry and membrane growth suggests a novel model for cell size control

PubMed Central

Anastasia, Steph D.; Nguyen, Duy Linh; Thai, Vu; Meloy, Melissa; MacDonough, Tracy

2012-01-01

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2ACdc55). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function. PMID:22451696

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

PubMed

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

PubMed Central

Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

2015-01-01

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

A link between mitotic entry and membrane growth suggests a novel model for cell size control.

PubMed

Anastasia, Steph D; Nguyen, Duy Linh; Thai, Vu; Meloy, Melissa; MacDonough, Tracy; Kellogg, Douglas R

2012-04-02

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function.

Integrated strain array for cellular mechanobiology studies

NASA Astrophysics Data System (ADS)

Simmons, C. S.; Sim, J. Y.; Baechtold, P.; Gonzalez, A.; Chung, C.; Borghi, N.; Pruitt, B. L.

2011-05-01

We have developed an integrated strain array for cell culture enabling high-throughput mechano-transduction studies. Biocompatible cell culture chambers were integrated with an acrylic pneumatic compartment and microprocessor-based control system. Each element of the array consists of a deformable membrane supported by a cylindrical pillar within a well. For user-prescribed waveforms, the annular region of the deformable membrane is pulled into the well around the pillar under vacuum, causing the pillar-supported region with cultured cells to be stretched biaxially. The optically clear device and pillar-based mechanism of operation enables imaging on standard laboratory microscopes. Straightforward fabrication utilizes off-the-shelf components, soft lithography techniques in polydimethylsiloxane and laser ablation of acrylic sheets. Proof of compatibility with basic biological assays and standard imaging equipment were accomplished by straining C2C12 skeletal myoblasts on the device for 6 h. At higher strains, cells and actin stress fibers realign with a circumferential preference.

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

PubMed Central

Stout, A. L.; Axelrod, D.

1994-01-01

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes. Images FIGURE 2 FIGURE 3 FIGURE 4 PMID:7811947

[The mass-spectrometry studies of the interaction of polyhexamethyleneguanidine with lipids].

PubMed

Lysytsia, A V; Rebriiev, A V

2014-01-01

In this work the integral components of the cytoplasmic membrane, lecithin and cholesterol were used for mass spectrometry analysis carried out on polyhexamethyleneguanidine (PHMG) mixtures with lipids. The study was performed by mass-spectrometry methods of the MALDI-TOF MS. Our results showed that despite the common use of PHGM polymer derivatives as disinfectants the persistent intermolecular complexes of PHMG oligomers with lipids were not formed. The binding of polycation PHMG with the membrane has been explained by the model proposed. According to this model PHGM can adhere to negatively charged plasma membrane of bacterial cell due to electrostatic interaction and the formation of loop-like structures. Similar stereochemistry mechanism makes the adsorption of the investigated polycation to membrane robust. The mechanism described together with additional destructive factors provides a reasonable explanation for the PHMG induced damage of bacterial cell plasma membrane and the biocide action of disinfectants prepared on the basis of the PHMG salts.

Integrated optical design for highly dynamic laser beam shaping with membrane deformable mirrors

NASA Astrophysics Data System (ADS)

Pütsch, Oliver; Stollenwerk, Jochen; Loosen, Peter

2017-02-01

The utilization of membrane deformable mirrors has raised its importance in laser materials processing since they enable the generation of highly spatial and temporal dynamic intensity distributions for a wide field of applications. To take full advantage of these devices for beam shaping, the huge amount of degrees of freedom has to be considered and optimized already within the early stage of the optical design. Since the functionality of commercial available ray-tracing software has been mainly specialized on geometric dependencies and their optimization within constraints, the complex system characteristics of deformable mirrors cannot be sufficiently taken into account yet. The main reasons are the electromechanical interdependencies of electrostatic membrane deformable mirrors, namely saturation and mechanical clamping, that result in non-linear deformation. This motivates the development of an integrative design methodology. The functionality of the ray-tracing program ZEMAX is extended with a model of an electrostatic membrane mirror. This model is based on experimentally determined influence functions. Furthermore, software routines are derived and integrated that allow for the compilation of optimization criteria for the most relevant analytically describable beam shaping problems. In this way, internal optimization routines can be applied for computing the appropriate membrane deflection of the deformable mirror as well as for the parametrization of static optical components. The experimental verification of simulated intensity distributions demonstrates that the beam shaping properties can be predicted with a high degree of reliability and precision.

Integration of β-carotene molecules in small liposomes

NASA Astrophysics Data System (ADS)

Andreeva, Atanaska; Popova, Antoaneta

2010-11-01

The most typical feature of carotenoids is the long polyene chain with conjugated double bonds suggesting that they can serve as conductors of electrons, acting as ''molecular wires'', important elements in the molecular electronic devices. Carotenoids are essential components of photosynthetic systems, performing different functions as light harvesting, photoprotection and electron transfer. They act also as natural antioxidants. In addition they perform structural role stabilizing the three-dimensional organization of photosynthetic membranes. Carotenoids contribute to the stability of the lipid phase, preserving the membrane integrity under potentially harmful environmental conditions. Carotenoids can be easily integrated into model membranes, facilitating the investigation of their functional roles. In carotenoid-egg phosphatidylcholine (EPC) liposomes ß-carotene is randomly distributed in the hydrocarbon interior of the bilayer, without any preferred, well defined orientation and retains a substantial degree of mobility. Here we investigate the degree of integration of ß-carotene in small unilamellar EPC liposomes and the changes in ß-carotene absorption and Raman spectra due to the lipid-pigment interaction. All observed changes in ß-carotene absorption and Raman spectra may be regarded as a result of the lipid-pigment interactions leading to the polyene geometry distortion and increasing of the environment heterogenety in the liposomes as compared to the solutions.

Electrogenesis in the lower Metazoa and implications for neuronal integration

PubMed Central

Meech, Robert W.

2015-01-01

Electrogenic communication appears to have evolved independently in a variety of animal and plant lineages. Considered here are metazoan cells as disparate as the loose three-dimensional parenchyma of glass sponges, the two-dimensional epithelial sheets of hydrozoan jellyfish and the egg cell membranes of the ctenophore Beroe ovata, all of which are capable of generating electrical impulses. Neuronal electrogenesis may have evolved independently in ctenophores and cnidarians but the dearth of electrophysiological data relating to ctenophore nerves means that our attention is focused on the Cnidaria, whose nervous systems have been the subject of extensive study. The aim here is to show how their active and passive neuronal properties interact to give integrated behaviour. Neuronal electrogenesis, goes beyond simply relaying ‘states of excitement’ and utilizes the equivalent of a set of basic electrical ‘apps’ to integrate incoming sensory information with internally generated pacemaker activity. A small number of membrane-based processes make up these analogue applications. Passive components include the decremental spread of current determined by cellular anatomy; active components include ion channels specified by their selectivity and voltage dependence. A recurring theme is the role of inactivating potassium channels in regulating performance. Although different aspects of cnidarian behaviour are controlled by separate neuronal systems, integrated responses and coordinated movements depend on interactions between them. Integrative interactions discussed here include those between feeding and swimming, between tentacle contraction and swimming and between slow and fast swimming in the hydrozoan jellyfish Aglantha digitale. PMID:25696817

Integrity of the Linker of Nucleoskeleton and Cytoskeleton Is Required for Efficient Herpesvirus Nuclear Egress.

PubMed

Klupp, Barbara G; Hellberg, Teresa; Granzow, Harald; Franzke, Kati; Dominguez Gonzalez, Beatriz; Goodchild, Rose E; Mettenleiter, Thomas C

2017-10-01

Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM. IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking. Copyright © 2017 American Society for Microbiology.

Caterpillar locomotion-inspired valveless pneumatic micropump using a single teardrop-shaped elastomeric membrane.

PubMed

So, Hongyun; Pisano, Albert P; Seo, Young Ho

2014-07-07

This paper presents a microfluidic pump operated by an asymmetrically deformed membrane, which was inspired by caterpillar locomotion. Almost all mechanical micropumps consist of two major components of fluid halting and fluid pushing parts, whereas the proposed caterpillar locomotion-inspired micropump has only a single, bilaterally symmetric membrane-like teardrop shape. A teardrop-shaped elastomeric membrane was asymmetrically deformed and then consecutively touched down to the bottom of the chamber in response to pneumatic pressure, thus achieving fluid pushing. Consecutive touchdown motions of the teardrop-shaped membrane mimicked the propagation of a caterpillar's hump during its locomotory gait. The initial touchdown motion of the teardrop-shaped membrane at the centroid worked as a valve that blocked the inlet channel, and then, the consecutive touchdown motions pushed fluid in the chamber toward the tail of the chamber connected to the outlet channel. The propagation of the touchdown motion of the teardrop-shaped membrane was investigated using computational analysis as well as experimental studies. This caterpillar locomotion-inspired micropump composed of only a single membrane can provide new opportunities for simple integration of microfluidic systems.

[Application on food preservative of antimicrobial peptides].

PubMed

Zhao, Hongyan; Mu, Yu; Zhao, Baohua

2009-07-01

Antimicrobial peptides are an integral component of the innate immune system, it can counteract outer membrane pathogen such as bacteria, fungi, viruses, protozoan and so on. Owing to the sterilization and innocuity, it has the potential to be crude food preservative. In this paper the uses of antibacterial peptides in the food preservative were analyzed.

Structural basis for catalysis at the membrane-water interface.

PubMed

Dufrisne, Meagan Belcher; Petrou, Vasileios I; Clarke, Oliver B; Mancia, Filippo

2017-11-01

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Elevated 2,3-diphosphoglycerate concentrations and alteration of structural integrity in the erythrocytes of Indian cases of visceral leishmaniasis.

PubMed

Biswas, T; Ghosh, D K; Mukherjee, N; Ghosal, J

1995-08-01

The visceral leishmaniasis (VL) known as kala-azar in India is characterized by severe anaemia. The anaemia seems to be the result, at least in part, of the relatively short life-time of the erythrocytes, which have weakened cell membranes, possibly because of elevated concentrations of 2,3-diphosphoglycerate (2,3-DPG). There is a negative correlation (r = 0.91; P < 0.01) between erythrocytic 2,3-DPG concentrations and the blood concentration of haemoglobin, and the erythrocytes from infected patients display higher osmotic fragility than those from uninfected controls. Spectrofluorometry, using 1,6-diphenyl 1,3,5-hexatriene as a probe, indicated that fluorescence depolarization and microviscosity are also higher in the erythrocytic membranes from VL cases than in those from the controls. The cholesterol/phospholipid ratio is also relatively high in the membranes from the VL cases and there is degradation of the skeletal components and the major integral protein (band 3). The enhanced concentration of 2,3-DPG may be related to the altered structural integrity of the erythrocytes and this may lead to anisocytosis and the reduction in the erythrocytic half life.

Influence of silica matrix composition and functional component additives on the bioactivity and viability of encapsulated living cells

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

Savage, Travis J.; Dunphy, Darren R.; Harbaugh, Svetlana

The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cellsmore » encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. Furthermore, this unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.« less

Influence of silica matrix composition and functional component additives on the bioactivity and viability of encapsulated living cells

DOE PAGES

Savage, Travis J.; Dunphy, Darren R.; Harbaugh, Svetlana; ...

2015-11-06

The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cellsmore » encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. Furthermore, this unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.« less

Membrane mimetic surface functionalization of nanoparticles: Methods and applications

PubMed Central

Weingart, Jacob; Vabbilisetty, Pratima; Sun, Xue-Long

2013-01-01

Nanoparticles (NPs), due to their size-dependent physical and chemical properties, have shown remarkable potential for a wide range of applications over the past decades. Particularly, the biological compatibilities and functions of NPs have been extensively studied for expanding their potential in areas of biomedical application such as bioimaging, biosensing, and drug delivery. In doing so, surface functionalization of NPs by introducing synthetic ligands and/or natural biomolecules has become a critical component in regards to the overall performance of the NP system for its intended use. Among known examples of surface functionalization, the construction of an artificial cell membrane structure, based on phospholipids, has proven effective in enhancing biocompatibility and has become a viable alternative to more traditional modifications, such as direct polymer conjugation. Furthermore, certain bioactive molecules can be immobilized onto the surface of phospholipid platforms to generate displays more reminiscent of cellular surface components. Thus, NPs with membrane-mimetic displays have found use in a range of bioimaging, biosensing, and drug delivery applications. This review herein describes recent advances in the preparations and characterization of integrated functional NPs covered by artificial cell membrane structures and their use in various biomedical applications. PMID:23688632

Cholesterol depletion in adipocytes causes caveolae collapse concomitant with proteosomal degradation of cavin-2 in a switch-like fashion.

PubMed

Breen, Michael R; Camps, Marta; Carvalho-Simoes, Francisco; Zorzano, Antonio; Pilch, Paul F

2012-01-01

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity.

Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion

PubMed Central

Breen, Michael R.; Camps, Marta; Carvalho-Simoes, Francisco; Zorzano, Antonio; Pilch, Paul F.

2012-01-01

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity. PMID:22493697

The transport along membrane nanotubes driven by the spontaneous curvature of membrane components.

PubMed

Kabaso, Doron; Bobrovska, Nataliya; Góźdź, Wojciech; Gongadze, Ekaterina; Kralj-Iglič, Veronika; Zorec, Robert; Iglič, Aleš

2012-10-01

Intercellular membrane nanotubes (ICNs) serve as a very specific transport system between neighboring cells. The underlying mechanisms responsible for the transport of membrane components and vesicular dilations along the ICNs are not clearly understood. The present study investigated the spatial distribution of anisotropic membrane components of tubular shapes and isotropic membrane components of spherical shapes. Experimental results revealed the preferential distribution of CTB (cholera toxin B)-GM1 complexes mainly on the spherical cell membrane, and cholesterol-sphingomyelin at the membrane leading edge and ICNs. In agreement with previous studies, we here propose that the spatial distribution of CTB-GM1 complexes and cholesterol-sphingomyelin rafts were due to their isotropic and anisotropic shapes, respectively. To elucidate the relationship between a membrane component shape and its spatial distribution, a two-component computational model was constructed. The minimization of the membrane bending (free) energy revealed the enrichment of the anisotropic component along the ICN and the isotropic component in the parent cell membrane, which was due to the curvature mismatch between the ICN curvature and the spontaneous curvature of the isotropic component. The equations of motion, derived from the differentiation of the membrane free energy, revealed a curvature-dependent flux of the isotropic component and a curvature-dependent force exerted on a vesicular dilation along the ICN. Finally, the effects of possible changes in the orientational ordering of the anisotropic component attendant to the transport of the vesicular dilation were discussed with connection to the propagation of electrical and chemical signals. Copyright © 2012 Elsevier B.V. All rights reserved.

Studying lipid-protein interactions with electron paramagnetic resonance spectroscopy of spin-labeled lipids.

PubMed

Páli, Tibor; Kóta, Zoltán

2013-01-01

Spin label electron paramagnetic resonance (EPR) of lipid-protein interactions reveals crucial features of the structure and assembly of integral membrane proteins. Spin label EPR spectroscopy is the technique of choice to characterize the protein-solvating lipid shell in its highly dynamic nature, because the EPR spectra of lipids that are spin labeled close to the terminal methyl end of their acyl chains display two spectral components, those corresponding to lipids directly contacting the protein and those corresponding to lipids in the bulk fluid bilayer regions of the membrane. In this chapter, typical spin label EPR procedures are presented that allow determination of the stoichiometry of interaction of spin-labeled lipids with the intra-membranous region of membrane proteins or polypeptides, as well as the association constant of the spin-labeled lipid with respect to the host lipid. The lipids giving rise to the so-called immobile spectral component in the EPR spectrum of such samples are identified as the motionally restricted first-shell lipids solvating membrane proteins in biomembranes. Stoichiometry and selectivity are directly related to the structure of the intra-membranous sections of membrane-associated proteins or polypeptides and can be used to study the state of assembly of such proteins in the membrane. Since these characteristics of lipid-protein interactions are discussed in detail in the literature [see Marsh (Eur Biophys J 39:513-525, 2010) for a most recent review], here we focus more on how to spin label model and biomembranes and how to measure and analyze the two-component EPR spectra of spin-labeled lipids in phospholipid bilayers that contain proteins or polypeptides. After a description of how to prepare spin-labeled model and native biological membranes, we present the reader with computational procedures for determining the molar fraction of motionally restricted lipids when both, one, or none of the pure isolated-mobile or immobile-spectral components are available. With these topics, this chapter complements a recent methodological paper [Marsh (Methods 46:83-96, 2008)]. The interpretation of the data is discussed briefly, as well as other relevant and recent spin label EPR techniques for studying lipid-protein interactions, not only from the point of view of lipid chain dynamics.

Membrane-Based Emitter for Coupling Microfluidics with Ultrasensitive Nanoelectrospray Ionization-Mass Spectrometry

PubMed Central

Sun, Xuefei; Kelly, Ryan T.; Tang, Keqi; Smith, Richard D.

2011-01-01

An integrated poly(dimethylsiloxane) (PDMS) membrane-based microfluidic emitter for high performance nanoelectrospray ionization-mass spectrometry (nanoESI-MS) has been fabricated and evaluated. The ~100-μm-thick emitter was created by cutting a PDMS membrane that protrudes beyond the bulk substrate. The reduced surface area at the emitter enhances the electric field and reduces wetting of the surface by the electrospray solvent. As such, the emitter enables highly stable electrosprays at flow rates as low as 10 nL/min, and is compatible with electrospray solvents containing a large organic component (e.g., 90% methanol). This approach enables facile emitter construction, and provides excellent stability, reproducibility and sensitivity, as well as compatibility with multilayer soft lithography. PMID:21657269

Controlling Cargo Trafficking in Multicomponent Membranes.

PubMed

Curk, Tine; Wirnsberger, Peter; Dobnikar, Jure; Frenkel, Daan; Šarić, Anđela

2018-04-27

Biological membranes typically contain a large number of different components dispersed in small concentrations in the main membrane phase, including proteins, sugars, and lipids of varying geometrical properties. Most of these components do not bind the cargo. Here, we show that such "inert" components can be crucial for the precise control of cross-membrane trafficking. Using a statistical mechanics model and molecular dynamics simulations, we demonstrate that the presence of inert membrane components of small isotropic curvatures dramatically influences cargo endocytosis, even if the total spontaneous curvature of such a membrane remains unchanged. Curved lipids, such as cholesterol, as well as asymmetrically included proteins and tethered sugars can, therefore, actively participate in the control of the membrane trafficking of nanoscopic cargo. We find that even a low-level expression of curved inert membrane components can determine the membrane selectivity toward the cargo size and can be used to selectively target membranes of certain compositions. Our results suggest a robust and general method of controlling cargo trafficking by adjusting the membrane composition without needing to alter the concentration of receptors or the average membrane curvature. This study indicates that cells can prepare for any trafficking event by incorporating curved inert components in either of the membrane leaflets.

Low-pressure membrane integrity tests for drinking water treatment: A review.

PubMed

Guo, H; Wyart, Y; Perot, J; Nauleau, F; Moulin, P

2010-01-01

Low-pressure membrane systems, including microfiltration (MF) and ultrafiltration (UF) membranes, are being increasingly used in drinking water treatments due to their high level of pathogen removal. However, the pathogen will pass through the membrane and contaminate the product if the membrane integrity is compromised. Therefore, an effective on-line integrity monitoring method for MF and UF membrane systems is essential to guarantee the regulatory requirements for pathogen removal. A lot of works on low-pressure membrane integrity tests have been conducted by many researchers. This paper provides a literature review about different low-pressure membrane integrity monitoring methods for the drinking water treatment, including direct methods (pressure-based tests, acoustic sensor test, liquid porosimetry, etc.) and indirect methods (particle counting, particle monitoring, turbidity monitoring, surrogate challenge tests). Additionally, some information about the operation of membrane integrity tests is presented here. It can be realized from this review that it remains urgent to develop an alternative on-line detection technique for a quick, accurate, simple, continuous and relatively inexpensive evaluation of low-pressure membrane integrity. To better satisfy regulatory requirements for drinking water treatments, the characteristic of this ideal membrane integrity test is proposed at the end of this paper.

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex 1

PubMed Central

Leto, Kenneth J.; Keresztes, Aron; Arntzen, Charles J.

1982-01-01

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [35S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts. Images PMID:16662421

Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

PubMed Central

Desvaux, Mickaël; Candela, Thomas; Serror, Pascale

2018-01-01

The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria) is formed of a cytoplasmic membrane (CM) and a cell wall (CW). While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG) covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO) for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226), i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658), i.e., the lipoproteins. At the CW (GO: 0009275), cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed. PMID:29491848

Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

NASA Astrophysics Data System (ADS)

Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

2014-07-01

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

Crystallographic snapshot of cellulose synthesis and membrane translocation.

PubMed

Morgan, Jacob L W; Strumillo, Joanna; Zimmer, Jochen

2013-01-10

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

PubMed

Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2018-06-27

The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

High-Force Dielectric Electroactive Polymer (DEAP) membrane actuator

NASA Astrophysics Data System (ADS)

Hau, Steffen; York, Alexander; Seelecke, Stefan

2016-04-01

Energy efficiency, lightweight and scalability are key features for actuators in applications such as valves, pumps or any portable system. Dielectric electroactive Polymer (DEAP) technology is able to fulfill these requirements1 better than commonly used technology e.g. solenoids, but has limitations concerning force and stroke. However, the circular DEAP membrane actuator shows a potential increase in stroke in the mm range, when combined with an appropriate biasing mechanism2. Although, thus far, their force range is limited to the single-digit Newton range, or less3,4. This work describes how this force limit of DEAP membrane actuators can be pushed to the high double-digit Newton range and beyond. The concept for such an actuator consists of a stack of double-layered DEAPs membrane actuator combined with a biasing mechanism. These two components are combined in a novel way, which allows a compact design by integrating the biasing mechanism into the DEAP membrane actuator stack. Subsequently, the single components are manufactured, tested, and their force-displacement characteristic is documented. Utilizing this data allows assembling them into actuator systems for different applications. Two different actuators are assembled and tested (dimensions: 85x85x30mm3 (LxWxH)). The first one is able to lift 7.5kg. The second one can generate a force of 66N while acting against a spring load.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

PubMed

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-04-28

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

PubMed Central

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-01-01

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo. However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. PMID:28283569

Biosynthesis of plant cell wall polysaccharides.

PubMed

Gibeaut, D M; Carpita, N C

1994-09-01

The cell wall is the principal structural element of plant form. Cellulose, long crystals of several dozen glucan chains, forms the microfibrillar foundation of plant cell walls and is synthesized at the plasma membrane. Except for callose, all other noncellulosic components are secreted to the cell surface and form a porous matrix assembled around the cellulose microfibrils. These diverse noncellulosic polysaccharides and proteins are made in the endomembrane system. Many questions about the biosynthesis and modification within the Golgi apparatus and integration of cell components at the cell surface remain unanswered. The lability of synthetic complexes upon isolation is one reason for slow progress. However, with new methods of membrane isolation and analysis of products in vitro, recent advances have been made in purifying active synthases from plasma membrane and Golgi apparatus. Likely synthase polypeptides have been identified by affinity-labeling techniques, but we are just beginning to understand the unique features of the coordinated assembly of complex polysaccharides. Nevertheless, such progress renews hope that the first gene of a synthase for a wall polysaccharide from higher plants is within our grasp.

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

Atomistic models for free energy evaluation of drug binding to membrane proteins.

PubMed

Durdagi, S; Zhao, C; Cuervo, J E; Noskov, S Y

2011-01-01

The binding of various molecules to integral membrane proteins with optimal affinity and specificity is central to normal function of cell. While membrane proteins represent about one third of the whole cell proteome, they are a majority of common drug targets. The quest for the development of computational models capable of accurate evaluation of binding affinities, decomposition of the binding into its principal components and thus mapping molecular mechanisms of binding remains one of the main goals of modern computational biophysics and related drug development. The primary scope of this review will be on the recent extension of computational methods for the study of drug binding to membrane proteins. Several examples of such applications will be provided ranging from secondary transporters to voltage gated channels. In this mini-review, we will provide a short summary on the breadth of different methods for binding affinity evaluation. These methods include molecular docking with docking scoring functions, molecular dynamics (MD) simulations combined with post-processing analysis using Molecular Mechanics/Poisson Boltzmann (Generalized Born) Surface Area (MM/PB(GB)SA), as well as direct evaluation of free energies from Free Energy Perturbation (FEP) with constraining schemes, and Potential of Mean Force (PMF) computations. We will compare advantages and shortcomings of popular techniques and provide discussion on the integrative strategies for drug development aimed at targeting membrane proteins.

Van der Waals interactions between planar substrate and tubular lipid membranes undergoing pearling instability

NASA Astrophysics Data System (ADS)

Valchev, G. S.; Djondjorov, P. A.; Vassilev, V. M.; Dantchev, D. M.

2017-10-01

In the current article we study the behavior of the van der Waals force between a planar substrate and an axisymmetric bilayer lipid membrane undergoing pearling instability, caused by uniform hydrostatic pressure difference. To do so, the recently suggested "surface integration approach" is used, which can be considered a generalization of the well known and widely used Derjaguin approximation. The static equilibrium shape after the occurrence of the instability is described in the framework of Helfrich's spontaneous curvature model. Some specific classes of exact analytical solutions to the corresponding shape equation are considered, and the components of the respective position vectors given in terms of elliptic integrals and Jacobi elliptic functions. The mutual orientation between the interacting objects is chosen such that the axis of revolution of the distorted cylinder be parallel to the plane bounding the substrate. Based on the discussed models and approaches we made some estimations for the studied force in real experimentally realizable systems, thus showing the possibility of pearling as an useful technique for reduction of the adhesion in variety of industrial processes using lipid membranes as carriers.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

PubMed

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar; Buck, Martin

2015-09-01

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. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response. Copyright © 2015 McDonald et al.

Membranes for redox flow battery applications.

PubMed

Prifti, Helen; Parasuraman, Aishwarya; Winardi, Suminto; Lim, Tuti Mariana; Skyllas-Kazacos, Maria

2012-06-19

The need for large scale energy storage has become a priority to integrate renewable energy sources into the electricity grid. Redox flow batteries are considered the best option to store electricity from medium to large scale applications. However, the current high cost of redox flow batteries impedes the wide spread adoption of this technology. The membrane is a critical component of redox flow batteries as it determines the performance as well as the economic viability of the batteries. The membrane acts as a separator to prevent cross-mixing of the positive and negative electrolytes, while still allowing the transport of ions to complete the circuit during the passage of current. An ideal membrane should have high ionic conductivity, low water intake and excellent chemical and thermal stability as well as good ionic exchange capacity. Developing a low cost, chemically stable membrane for redox flow cell batteries has been a major focus for many groups around the world in recent years. This paper reviews the research work on membranes for redox flow batteries, in particular for the all-vanadium redox flow battery which has received the most attention.

Cavin family proteins and the assembly of caveolae

PubMed Central

Kovtun, Oleksiy; Tillu, Vikas A.; Ariotti, Nicholas; Parton, Robert G.; Collins, Brett M.

2015-01-01

ABSTRACT Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein–protein and protein–lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins. PMID:25829513

Membranes for Redox Flow Battery Applications

PubMed Central

Prifti, Helen; Parasuraman, Aishwarya; Winardi, Suminto; Lim, Tuti Mariana; Skyllas-Kazacos, Maria

2012-01-01

The need for large scale energy storage has become a priority to integrate renewable energy sources into the electricity grid. Redox flow batteries are considered the best option to store electricity from medium to large scale applications. However, the current high cost of redox flow batteries impedes the wide spread adoption of this technology. The membrane is a critical component of redox flow batteries as it determines the performance as well as the economic viability of the batteries. The membrane acts as a separator to prevent cross-mixing of the positive and negative electrolytes, while still allowing the transport of ions to complete the circuit during the passage of current. An ideal membrane should have high ionic conductivity, low water intake and excellent chemical and thermal stability as well as good ionic exchange capacity. Developing a low cost, chemically stable membrane for redox flow cell batteries has been a major focus for many groups around the world in recent years. This paper reviews the research work on membranes for redox flow batteries, in particular for the all-vanadium redox flow battery which has received the most attention. PMID:24958177

Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.

PubMed

Maekawa, Masashi; Fairn, Gregory D

2015-04-01

Cholesterol is an essential component of metazoan cellular membranes and it helps to maintain the structural integrity and fluidity of the plasma membrane. Here, we developed a cholesterol biosensor, termed D4H, based on the fourth domain of Clostridium perfringens theta-toxin, which recognizes cholesterol in the cytosolic leaflet of the plasma membrane and organelles. The D4H probe disassociates from the plasma membrane upon cholesterol extraction and after perturbations in cellular cholesterol trafficking. When used in combination with a recombinant version of the biosensor, we show that plasmalemmal phosphatidylserine is essential for retaining cholesterol in the cytosolic leaflet of the plasma membrane. In vitro experiments reveal that 1-stearoy-2-oleoyl phosphatidylserine can induce phase separation in cholesterol-containing lipid bilayers and shield cholesterol from cholesterol oxidase. Finally, the altered transbilayer distribution of cholesterol causes flotillin-1 to relocalize to endocytic organelles. This probe should be useful in the future to study pools of cholesterol in the cytosolic leaflet of the plasma membrane and organelles. © 2015. Published by The Company of Biologists Ltd.

VP08R from Infectious Spleen and Kidney Necrosis Virus Is a Novel Component of the Virus-Mock Basement Membrane

PubMed Central

Xu, Xiaopeng; Yan, Muting; Wang, Rui; Lin, Ting; Tang, Junliang; Li, Chaozheng; Weng, Shaoping

2014-01-01

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement membrane (BM)-like structure, termed virus-mock basement membrane (VMBM), on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R is located on the plasma membrane of infected cells and shows an expression profile similar to that of VP23R. Coimmunoprecipitation showed that VP08R interacts with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in the maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV; genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct virus-mock basement membranes (VMBMs) on the surface of infected cells to provide attaching sites for LECs. Although VMBMs lack the collagen IV network, which is an essential structural part of true BMs, VMBMs still show an intact structure. An ISKNV-encoded VP08R protein can self-assemble into a multimer and bind both VP23R and nidogen-1 to maintain the integrity and stability of VMBMs. On the basis of these facts, we redrew the putative schematic illustration of the VMBM structure. Our study suggests that the virus adopts a strategy to remodel the cellular matrix and may provide an important reference to elucidate BM functions and the mechanisms of lymphangiogenesis. PMID:24599992

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Organic fluid permeation through fluoropolymer membranes

DOEpatents

Nemser, Stuart M.; Kosaraju, Praveen; Bowser, John

2015-07-14

Separation of the components of liquid mixtures is achieved by contacting a liquid mixture with a nonporous membrane having a fluoropolymer selectively permeable layer and imposing a pressure gradient across the membrane from feed side to permeate side. Unusually high transmembrane flux is obtained when the membrane is subjected to one or more process conditions prior to separation. These include (a) leaving some residual amount of membrane casting solvent in the membrane, and (b) contacting the membrane with a component of the mixture to be separated for a duration effective to saturate the membrane with the component.

Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

PubMed Central

Discher, D E; Mohandas, N

1996-01-01

Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:8889146

Influences of acid-base property of membrane on interfacial interactions related with membrane fouling in a membrane bioreactor based on thermodynamic assessment.

PubMed

Zhao, Leihong; Qu, Xiaolu; Zhang, Meijia; Lin, Hongjun; Zhou, Xiaoling; Liao, Bao-Qiang; Mei, Rongwu; Hong, Huachang

2016-08-01

Failure of membrane hydrophobicity in predicting membrane fouling requires a more reliable indicator. In this study, influences of membrane acid base (AB) property on interfacial interactions in two different interaction scenarios in a submerged membrane bioreactor (MBR) were studied according to thermodynamic approaches. It was found that both the polyvinylidene fluoride (PVDF) membrane and foulant samples in the MBR had relatively high electron donor (γ(-)) component and low electron acceptor (γ(+)) component. For both of interaction scenarios, AB interaction was the major component of the total interaction. The results showed that, the total interaction monotonically decreased with membrane γ(-), while was marginally affected by membrane γ(+), suggesting that γ(-) could act as a reliable indicator for membrane fouling prediction. This study suggested that membrane modification for fouling mitigation should orient to improving membrane surface γ(-) component rather than hydrophilicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Properties and degradation of the gasket component of a proton exchange membrane fuel cell--a review.

PubMed

Basuli, Utpal; Jose, Jobin; Lee, Ran Hee; Yoo, Yong Hwan; Jeong, Kwang-Un; Ahn, Jou-Hyeon; Nah, Changwoon

2012-10-01

Proton exchange membrane (PEM) fuel cell stack requires gaskets and seals in each cell to keep the reactant gases within their respective regions. Gasket performance is integral to the successful long-term operation of a fuel cell stack. This review focuses on properties, performance and degradation mechanisms of the different polymer gasket materials used in PEM fuel cell under normal operating conditions. The different degradation mechanisms and their corresponding representative mitigation strategies are also presented here. Summary of various properties of elastomers and their advantages and disadvantages in fuel cell'environment are presented. By considering the level of chemical degradation, mechanical properties and cost effectiveness, it can be proposed that EPDM is one of the best choices for gasket material in PEM fuel cell. Finally, the challenges that remain in using rubber component as in PEM fuel cell, as well as the prospects for exploiting them in the future are discussed.

Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex

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

Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy

2014-08-19

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133 55–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one constructmore » of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.« less

Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex*

PubMed Central

Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy; Martel, Anne; Matsui, Tsutomu; Tsuruta, Hiro; Weiss, Thomas M.; Shi, Yi; Markina-Inarrairaegui, Ane; Bonanno, Jeffery B.; Sauder, J. Michael; Burley, Stephen K.; Chait, Brian T.; Almo, Steven C.; Rout, Michael P.; Sali, Andrej

2014-01-01

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup13355–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. PMID:25139911

Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: a high resolution ultrastructural study.

PubMed

Inoue, S; Osmond, D G

2001-11-01

Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the exceptionally large quantity of CSPG may represent a reservoir of CD44 receptor for use in hemopoiesis. Copyright 2001 Wiley-Liss, Inc.

Heuristics for the Hodgkin-Huxley system.

PubMed

Hoppensteadt, Frank

2013-09-01

Hodgkin and Huxley (HH) discovered that voltages control ionic currents in nerve membranes. This led them to describe electrical activity in a neuronal membrane patch in terms of an electronic circuit whose characteristics were determined using empirical data. Due to the complexity of this model, a variety of heuristics, including relaxation oscillator circuits and integrate-and-fire models, have been used to investigate activity in neurons, and these simpler models have been successful in suggesting experiments and explaining observations. Connections between most of the simpler models had not been made clear until recently. Shown here are connections between these heuristics and the full HH model. In particular, we study a new model (Type III circuit): It includes the van der Pol-based models; it can be approximated by a simple integrate-and-fire model; and it creates voltages and currents that correspond, respectively, to the h and V components of the HH system. Copyright © 2012 Elsevier Inc. All rights reserved.

Spectrin tetramer-dimer equilibrium and the stability of erythrocyte membrane skeletons

NASA Astrophysics Data System (ADS)

Liu, Shih-Chun; Palek, Jiri

1980-06-01

The inner side of the red-cell membrane is laminated by a two-dimensional network of membrane proteins which include spectrin, actin and some other components1-4. After extraction of lipids and integral proteins from the membrane, this membrane skeleton can be visualized as a ball-shaped network consisting of twisted fibres1-4 and globular protrusions4; however, the assembly of the individual proteins in the membrane skeleton is not well understood. Spectrin can be eluted from the membrane in the form of dimers and tetramers5-8. Electron microscopic study with low-angle shadowing technique shows that spectrin dimers are two parallel strands of twisted fibres presumably representing bands 1 and 2 of spectrin9. Spectrin tetramers presumably formed by head-to-head associations of two dimers are twice as long9. In solution, the spectrin dimer-tetramer equilibrium depends on temperature and salt concentration7,8; however, it is not known whether the same equilibrium exists in the membrane and whether it affects the physical properties of the membrane, such as its structural stability and deformability. We now demonstrate that spectrin dimers and tetramers are in a reversible equilibrium in the membrane and that in physiological conditions this equilibrium favours spectrin tetramers. Furthermore, we show that transformation of spectrin tetramers to dimers, as induced by ghost incubation in hypotonic conditions, diminishes the structural stability of the Triton-insoluble membrane skeletons.

Analysis of truss, beam, frame, and membrane components. [composite structures

NASA Technical Reports Server (NTRS)

Knoell, A. C.; Robinson, E. Y.

1975-01-01

Truss components are considered, taking into account composite truss structures, truss analysis, column members, and truss joints. Beam components are discussed, giving attention to composite beams, laminated beams, and sandwich beams. Composite frame components and composite membrane components are examined. A description is given of examples of flat membrane components and examples of curved membrane elements. It is pointed out that composite structural design and analysis is a highly interactive, iterative procedure which does not lend itself readily to characterization by design or analysis function only.-

Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane.

PubMed

Ji, Hongtao; Dong, Hansong

2015-09-01

Many plant- and animal-pathogenic Gram-negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel-like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two-step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two-domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin-rich middle lamella by the bacterial pilus. One-domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin-type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing

PubMed Central

Xue, Rui; Liu, Yalong; Liang, Congcong; Qin, Huazhen; Liu, Pengfei; Wang, Ke; Zhang, Xiaoyong; Chen, Li

2016-01-01

ABSTRACT To verify the interaction mechanism between sericin and Escherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin against E. coli as a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin on E. coli and the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for an in vivo study of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing of E. coli cells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. When E. coli cells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction of E. coli. Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels. IMPORTANCE The specific relationship and interaction mechanism between sericin and E. coli cells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing of E. coli cells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequent in vivo results demonstrate that the sericin-poly(N-isopropylacrylamide-N,N′-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries. PMID:27235427

Dormancy and Recovery Testing for Biological Wastewater Processors

NASA Technical Reports Server (NTRS)

Hummerick, Mary F.; Coutts, Janelle L.; Lunn, Griffin M.; Spencer, LaShelle; Khodadad, Christina L.; Birmele, Michele N.; Frances, Someliz; Wheeler, Raymond

2015-01-01

Resource recovery and recycling waste streams to usable water via biological water processors is a plausible component of an integrated water purification system. Biological processing as a pretreatment can reduce the load of organic carbon and nitrogen compounds entering physiochemical systems downstream. Aerated hollow fiber membrane bioreactors, have been proposed and studied for a number of years as an approach for treating wastewater streams for space exploration.

Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

PubMed

Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P

2012-03-01

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. Copyright © 2011 Elsevier B.V. All rights reserved.

Membrane Lipid Peroxidation in Copper Alloy-Mediated Contact Killing of Escherichia coli

PubMed Central

Hong, Robert; Kang, Tae Y.; Michels, Corinne A.

2012-01-01

Copper alloy surfaces are passive antimicrobial sanitizing agents that kill bacteria, fungi, and some viruses. Studies of the mechanism of contact killing in Escherichia coli implicate the membrane as the target, yet the specific component and underlying biochemistry remain unknown. This study explores the hypothesis that nonenzymatic peroxidation of membrane phospholipids is responsible for copper alloy-mediated surface killing. Lipid peroxidation was monitored with the thiobarbituric acid-reactive substances (TBARS) assay. Survival, TBARS levels, and DNA degradation were followed in cells exposed to copper alloy surfaces containing 60 to 99.90% copper or in medium containing CuSO4. In all cases, TBARS levels increased with copper exposure levels. Cells exposed to the highest copper content alloys, C11000 and C24000, exhibited novel characteristics. TBARS increased immediately at a very rapid rate but peaked at about 30 min. This peak was associated with the period of most rapid killing, loss in membrane integrity, and DNA degradation. DNA degradation is not the primary cause of copper-mediated surface killing. Cells exposed to the 60% copper alloy for 60 min had fully intact genomic DNA but no viable cells. In a fabR mutant strain with increased levels of unsaturated fatty acids, sensitivity to copper alloy surface-mediated killing increased, TBARS levels peaked earlier, and genomic DNA degradation occurred sooner than in the isogenic parental strain. Taken together, these results suggest that copper alloy surface-mediated killing of E. coli is triggered by nonenzymatic oxidative damage of membrane phospholipids that ultimately results in the loss of membrane integrity and cell death. PMID:22247141

Mechanical membrane for the separation of a paramagnetic constituent from a fluid

DOEpatents

Maurice, David

2017-05-02

The disclosure provides an apparatus and method for the separation of a paramagnetic component from a mixture using a mechanical membrane apparatus. The mechanical membrane comprises a supporting material having a plurality of pores where each pore is surrounded by a plurality of magnetic regions. The magnetic regions augment a magnetic field on one side of the supporting material while mitigating the field to near zero on the opposite side. In operation, a flow of fluid such as air comprising a paramagnetic component such as O.sub.2 is directed toward the mechanical membrane, and the paramagnetic component is typically attracted toward a magnetic field surrounding a pore while dimagnetic components such as N.sub.2 are generally repelled. As some portion of the fluid passes through the plurality of magnetic apertures to the opposite side of the mechanical membrane, the mechanical membrane generates a fluid enriched in the paramagnetic component. Alternately, the magnetic field may act to repel the paramagnetic component while diamagnetic components such as N.sub.2 are generally unaffected and pass to the opposite side of the mechanical membrane.

Future directions of electron crystallography.

PubMed

Fujiyoshi, Yoshinori

2013-01-01

In biological science, there are still many interesting and fundamental yet difficult questions, such as those in neuroscience, remaining to be answered. Structural and functional studies of membrane proteins, which are key molecules of signal transduction in neural and other cells, are essential for understanding the molecular mechanisms of many fundamental biological processes. Technological and instrumental advancements of electron microscopy have facilitated comprehension of structural studies of biological components, such as membrane proteins. While X-ray crystallography has been the main method of structure analysis of proteins including membrane proteins, electron crystallography is now an established technique to analyze structures of membrane proteins in the lipid bilayer, which is close to their natural biological environment. By utilizing cryo-electron microscopes with helium-cooled specimen stages, structures of membrane proteins were analyzed at a resolution better than 3 Å. Such high-resolution structural analysis of membrane proteins by electron crystallography opens up the new research field of structural physiology. Considering the fact that the structures of integral membrane proteins in their native membrane environment without artifacts from crystal contacts are critical in understanding their physiological functions, electron crystallography will continue to be an important technology for structural analysis. In this chapter, I will present several examples to highlight important advantages and to suggest future directions of this technique.

Presenilins and γ-Secretase: Structure, Function, and Role in Alzheimer Disease

PubMed Central

De Strooper, Bart; Iwatsubo, Takeshi; Wolfe, Michael S.

2012-01-01

Presenilins were first discovered as sites of missense mutations responsible for early-onset Alzheimer disease (AD). The encoded multipass membrane proteins were subsequently found to be the catalytic components of γ-secretases, membrane-embedded aspartyl protease complexes responsible for generating the carboxyl terminus of the amyloid β-protein (Aβ) from the amyloid protein precursor (APP). The protease complex also cleaves a variety of other type I integral membrane proteins, most notably the Notch receptor, signaling from which is involved in many cell differentiation events. Although γ-secretase is a top target for developing disease-modifying AD therapeutics, interference with Notch signaling should be avoided. Compounds that alter Aβ production by γ-secretase without affecting Notch proteolysis and signaling have been identified and are currently at various stages in the drug development pipeline. PMID:22315713

Antimicrobial Activity and Possible Mechanism of Action of Citral against Cronobacter sakazakii.

PubMed

Shi, Chao; Song, Kaikuo; Zhang, Xiaorong; Sun, Yi; Sui, Yue; Chen, Yifei; Jia, Zhenyu; Sun, Huihui; Sun, Zheng; Xia, Xiaodong

2016-01-01

Citral is a flavor component that is commonly used in food, beverage and fragrance industries. Cronobacter sakazakii is a food-borne pathogen associated with severe illness and high mortality in neonates and infants. The objective of the present study was to evaluate antimicrobial effect of citral against C. sakazakii strains. The minimum inhibitory concentration (MIC) of citral against C. sakazakii was determined via agar dilution method, then Gompertz models were used to quantitate the effect of citral on microbial growth kinetics. Changes in intracellular pH (pHin), membrane potential, intracellular ATP concentration, and membrane integrity were measured to elucidate the possible antimicrobial mechanism. Cell morphology changes were also examined using a field emission scanning electron microscope. The MICs of citral against C. sakazakii strains ranged from 0.27 to 0.54 mg/mL, and citral resulted in a longer lag phase and lower growth rate of C. sakazakii compared to the control. Citral affected the cell membrane of C. sakazakii, as evidenced by decreased intracellular ATP concentration, reduced pHin, and cell membrane hyperpolarization. Scanning electron microscopy analysis further confirmed that C. sakazakii cell membranes were damaged by citral. These findings suggest that citral exhibits antimicrobial effect against C. sakazakii strains and could be potentially used to control C. sakazakii in foods. However, how it works in food systems where many other components may interfere with its efficacy should be tested in future research before its real application.

Antimicrobial Activity and Possible Mechanism of Action of Citral against Cronobacter sakazakii

PubMed Central

Shi, Chao; Song, Kaikuo; Zhang, Xiaorong; Sun, Yi; Sui, Yue; Chen, Yifei; Jia, Zhenyu; Sun, Huihui; Sun, Zheng; Xia, Xiaodong

2016-01-01

Citral is a flavor component that is commonly used in food, beverage and fragrance industries. Cronobacter sakazakii is a food-borne pathogen associated with severe illness and high mortality in neonates and infants. The objective of the present study was to evaluate antimicrobial effect of citral against C. sakazakii strains. The minimum inhibitory concentration (MIC) of citral against C. sakazakii was determined via agar dilution method, then Gompertz models were used to quantitate the effect of citral on microbial growth kinetics. Changes in intracellular pH (pHin), membrane potential, intracellular ATP concentration, and membrane integrity were measured to elucidate the possible antimicrobial mechanism. Cell morphology changes were also examined using a field emission scanning electron microscope. The MICs of citral against C. sakazakii strains ranged from 0.27 to 0.54 mg/mL, and citral resulted in a longer lag phase and lower growth rate of C. sakazakii compared to the control. Citral affected the cell membrane of C. sakazakii, as evidenced by decreased intracellular ATP concentration, reduced pHin, and cell membrane hyperpolarization. Scanning electron microscopy analysis further confirmed that C. sakazakii cell membranes were damaged by citral. These findings suggest that citral exhibits antimicrobial effect against C. sakazakii strains and could be potentially used to control C. sakazakii in foods. However, how it works in food systems where many other components may interfere with its efficacy should be tested in future research before its real application. PMID:27415761

Transport Studies and Modeling in PEM Fuel Cells

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

Mittelsteadt, Cortney K.; Xu, Hui; Brawn, Shelly

2014-07-30

This project’s aim was to develop fuel cell components (i.e. membranes, gas-diffusion media (GDM), bipolar plates and flow fields) that possess specific properties (i.e. water transport and conductivity). A computational fluid dynamics model was developed to elucidate the effect of certain parameters on these specific properties. Ultimately, the model will be used to determine sensitivity of fuel cell performance to component properties to determine limiting components and to guide research. We have successfully reached our objectives and achieved most of the milestones of this project. We have designed and synthesized a variety of hydrocarbon block polymer membranes with lower equivalentmore » weight, structure, chemistry, phase separation and process conditions. These membranes provide a broad selection with optimized water transport properties. We have also designed and constructed a variety of devices that are capable of accurately measuring the water transport properties (water uptake, water diffusivity and electro-osmatic drag) of these membranes. These transport properties are correlated to the membranes’ structures derived from X-ray and microscopy techniques to determine the structure-property relationship. We successfully integrated hydrocarbon membrane MEAs with a current distribution board (CBD) to study the impact of hydrocarbon membrane on water transport in fuel cells. We have designed and fabricated various GDM with varying substrate, diffusivity and micro-porous layers (MPL) and characterized their pore structure, tortuosity and hydrophobicity. We have derived a universal chart (MacMullin number as function of wet proofing and porosity) that can be used to characterize various GDM. The abovementioned GDMs have been evaluated in operating fuel cells; their performance is correlated to various pore structure, tortuosity and hydrophobicity of the GDM. Unfortunately, determining a universal relationship between the MacMullin number and these properties was not achieved. We have simulated fuel cell performance, current distribution and water distribution at various values of the water uptake, membrane diffusivity, and electro-osmotic drag coefficient (EODC) and compared modeling results with segmented-cell data for both serpentine and parallel flow-fields. We have developed iterations of fuel cell flow fields to achieve specific water transport and thermal management targets. This work demonstrated the importance of membrane diffusivity on fuel cell performance, the necessity of a high membrane diffusion coefficient, and the desirability of a low EODC at low levels of relative humidity.« less

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

PubMed

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

2015-01-01

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

Functionalized Vesicles by Microfluidic Device.

PubMed

Vallejo, Derek; Lee, Shih-Hui; Lee, Abraham

2017-01-01

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.

Membrane pore architecture of the CslF6 protein controls (1-3,1-4)-β-glucan structure.

PubMed

Jobling, Stephen A

2015-06-01

The cereal cell wall polysaccharide (1-3,1-4)-β-glucan is a linear polymer of glucose containing both β1-3 and β1-4 bonds. The structure of (1-3,1-4)-β-glucan varies between different cereals and during plant growth and development, but little is known about how this is controlled. The cellulose synthase-like CslF6 protein is an integral membrane protein and a major component of the (1-3,1-4)-β-glucan synthase. I show that a single amino acid within the predicted transmembrane pore domain of CslF6 controls (1-3,1-4)-β-glucan structure. A new mechanism for the control of the polysaccharide structure is proposed where membrane pore architecture and the translocation of the growing polysaccharide across the membrane control how the acceptor glucan is coordinated at the active site and thus the proportion of β1-3 and β1-4 bonds within the polysaccharide.

Design of a homogeneous multifunctional supported lipid membrane on layer-by-layer coated microcarriers.

PubMed

Göse, Martin; Pescador, Paula; Reibetanz, Uta

2015-03-09

Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on a HAB causing Alexandrium tamarense.

PubMed

Li, Yi; Zhu, Hong; Zhang, Huajun; Chen, Zhangran; Tian, Yun; Xu, Hong; Zheng, Tianling; Zheng, Wei

2014-08-15

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on Alexandrium tamarense were measured through studying the algicidal procedure, nuclear damage and transcription of related genes. Medium components were optimized to improve algicidal activity, and characteristics of algicidal extracts were determined. Transmission electron microscope analysis revealed that the cell structure was broken. Cell membrane integrity destruction and nuclear structure degradation were monitored using confocal laser scanning microscope, and the rbcS, hsp and proliferating cell nuclear antigen (PCNA) gene expressions were studied. Results showed that 1.0% tryptone, 0.4% glucose and 0.8% MgCl2 were the optimal nutrient sources. The algicidal extracts were heat and pH stable, non-protein and less than 1kD. Cell membrane and nuclear structure integrity were lost, and the transcription of the rbcS and PCNA genes were significantly inhibited and there was up-regulation of hsp gene expression during the exposure procedure. The algicidal extracts destroyed the cell membrane and nuclear structure integrity, inhibited related gene expression and, eventually, lead to the inhibition of algal growth. All the results may elaborate firstly the cell death process and nuclear damage in A. tamarense which was induced by algicidal extracts, and the algicidal extracts could be potentially used as bacterial control of HABs in future. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural Engineering for High Sensitivity, Ultrathin Pressure Sensors Based on Wrinkled Graphene and Anodic Aluminum Oxide Membrane.

PubMed

Chen, Wenjun; Gui, Xuchun; Liang, Binghao; Yang, Rongliang; Zheng, Yongjia; Zhao, Chengchun; Li, Xinming; Zhu, Hai; Tang, Zikang

2017-07-19

Nature-motivated pressure sensors have been greatly important components integrated into flexible electronics and applied in artificial intelligence. Here, we report a high sensitivity, ultrathin, and transparent pressure sensor based on wrinkled graphene prepared by a facile liquid-phase shrink method. Two pieces of wrinkled graphene are face to face assembled into a pressure sensor, in which a porous anodic aluminum oxide (AAO) membrane with the thickness of only 200 nm was used to insulate the two layers of graphene. The pressure sensor exhibits ultrahigh operating sensitivity (6.92 kPa -1 ), resulting from the insulation in its inactive state and conduction under compression. Formation of current pathways is attributed to the contact of graphene wrinkles through the pores of AAO membrane. In addition, the pressure sensor is also an on/off and energy saving device, due to the complete isolation between the two graphene layers when the sensor is not subjected to any pressure. We believe that our high-performance pressure sensor is an ideal candidate for integration in flexible electronics, but also paves the way for other 2D materials to be involved in the fabrication of pressure sensors.

A novel integrated thermal-/membrane-based solar energy-driven hybrid desalination system: Concept description and simulation results.

PubMed

Kim, Young-Deuk; Thu, Kyaw; Ng, Kim Choon; Amy, Gary L; Ghaffour, Noreddine

2016-09-01

In this paper, a hybrid desalination system consisting of vacuum membrane distillation (VMD) and adsorption desalination (AD) units, designated as VMD-AD cycle, is proposed. The synergetic integration of the VMD and AD is demonstrated where a useful effect of the AD cycle is channelled to boost the operation of the VMD process, namely the low vacuum environment to maintain the high pressure gradient across the microporous hydrophobic membrane. A solar-assisted multi-stage VMD-AD hybrid desalination system with temperature modulating unit is first designed, and its performance is then examined with a mathematical model of each component in the system and compared with the VMD-only system with temperature modulating and heat recovery units. The total water production and water recovery ratio of a solar-assisted 24-stage VMD-AD hybrid system are found to be about 21% and 23% higher, respectively, as compared to the VMD-only system. For the solar-assisted 24-stage VMD-AD desalination system having 150 m(2) of evacuated-tube collectors and 10 m(3) seawater storage tanks, both annual collector efficiency and solar fraction are close to 60%. Copyright © 2016 Elsevier Ltd. All rights reserved.

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

PubMed Central

Diaz, Maira; Sanchez-Barrena, Maria Jose; Gonzalez-Rubio, Juana Maria; Rodriguez, Lesia; Fernandez, Daniel; Antoni, Regina; Yunta, Cristina; Belda-Palazon, Borja; Gonzalez-Guzman, Miguel; Peirats-Llobet, Marta; Menendez, Margarita; Boskovic, Jasminka; Marquez, Jose A.; Rodriguez, Pedro L.; Albert, Armando

2016-01-01

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca2+-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca2+-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress. PMID:26719420

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling.

PubMed

Diaz, Maira; Sanchez-Barrena, Maria Jose; Gonzalez-Rubio, Juana Maria; Rodriguez, Lesia; Fernandez, Daniel; Antoni, Regina; Yunta, Cristina; Belda-Palazon, Borja; Gonzalez-Guzman, Miguel; Peirats-Llobet, Marta; Menendez, Margarita; Boskovic, Jasminka; Marquez, Jose A; Rodriguez, Pedro L; Albert, Armando

2016-01-19

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.

Release of outer membrane vesicles from Bordetella pertussis.

PubMed

Hozbor, D; Rodriguez, M E; Fernández, J; Lagares, A; Guiso, N; Yantorno, O

1999-05-01

The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly- showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

Molecular dynamics simulations of heterogeneous cell membranes in response to uniaxial membrane stretches at high loading rates.

PubMed

Zhang, Lili; Zhang, Zesheng; Jasa, John; Li, Dongli; Cleveland, Robin O; Negahban, Mehrdad; Jérusalem, Antoine

2017-08-16

The chemobiomechanical signatures of diseased cells are often distinctively different from that of healthy cells. This mainly arises from cellular structural/compositional alterations induced by disease development or therapeutic molecules. Therapeutic shock waves have the potential to mechanically destroy diseased cells and/or increase cell membrane permeability for drug delivery. However, the biomolecular mechanisms by which shock waves interact with diseased and healthy cellular components remain largely unknown. By integrating atomistic simulations with a novel multiscale numerical framework, this work provides new biomolecular mechanistic perspectives through which many mechanosensitive cellular processes could be quantitatively characterised. Here we examine the biomechanical responses of the chosen representative membrane complexes under rapid mechanical loadings pertinent to therapeutic shock wave conditions. We find that their rupture characteristics do not exhibit significant sensitivity to the applied strain rates. Furthermore, we show that the embedded rigid inclusions markedly facilitate stretch-induced membrane disruptions while mechanically stiffening the associated complexes under the applied membrane stretches. Our results suggest that the presence of rigid molecules in cellular membranes could serve as "mechanical catalysts" to promote the mechanical destructions of the associated complexes, which, in concert with other biochemical/medical considerations, should provide beneficial information for future biomechanical-mediated therapeutics.

Materials and characterization techniques for high-temperature polymer electrolyte membrane fuel cells.

PubMed

Zeis, Roswitha

2015-01-01

The performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFC) is critically dependent on the selection of materials and optimization of individual components. A conventional high-temperature membrane electrode assembly (HT-MEA) primarily consists of a polybenzimidazole (PBI)-type membrane containing phosphoric acid and two gas diffusion electrodes (GDE), the anode and the cathode, attached to the two surfaces of the membrane. This review article provides a survey on the materials implemented in state-of-the-art HT-MEAs. These materials must meet extremely demanding requirements because of the severe operating conditions of HT-PEMFCs. They need to be electrochemically and thermally stable in highly acidic environment. The polymer membranes should exhibit high proton conductivity in low-hydration and even anhydrous states. Of special concern for phosphoric-acid-doped PBI-type membranes is the acid loss and management during operation. The slow oxygen reduction reaction in HT-PEMFCs remains a challenge. Phosphoric acid tends to adsorb onto the surface of the platinum catalyst and therefore hampers the reaction kinetics. Additionally, the binder material plays a key role in regulating the hydrophobicity and hydrophilicity of the catalyst layer. Subsequently, the binder controls the electrode-membrane interface that establishes the triple phase boundary between proton conductive electrolyte, electron conductive catalyst, and reactant gases. Moreover, the elevated operating temperatures promote carbon corrosion and therefore degrade the integrity of the catalyst support. These are only some examples how materials properties affect the stability and performance of HT-PEMFCs. For this reason, materials characterization techniques for HT-PEMFCs, either in situ or ex situ, are highly beneficial. Significant progress has recently been made in this field, which enables us to gain a better understanding of underlying processes occurring during fuel cell operation. Various novel tools for characterizing and diagnosing HT-PEMFCs and key components are presented in this review, including FTIR and Raman spectroscopy, confocal Raman microscopy, synchrotron X-ray imaging, X-ray microtomography, and atomic force microscopy.

Materials and characterization techniques for high-temperature polymer electrolyte membrane fuel cells

PubMed Central

2015-01-01

Summary The performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFC) is critically dependent on the selection of materials and optimization of individual components. A conventional high-temperature membrane electrode assembly (HT-MEA) primarily consists of a polybenzimidazole (PBI)-type membrane containing phosphoric acid and two gas diffusion electrodes (GDE), the anode and the cathode, attached to the two surfaces of the membrane. This review article provides a survey on the materials implemented in state-of-the-art HT-MEAs. These materials must meet extremely demanding requirements because of the severe operating conditions of HT-PEMFCs. They need to be electrochemically and thermally stable in highly acidic environment. The polymer membranes should exhibit high proton conductivity in low-hydration and even anhydrous states. Of special concern for phosphoric-acid-doped PBI-type membranes is the acid loss and management during operation. The slow oxygen reduction reaction in HT-PEMFCs remains a challenge. Phosphoric acid tends to adsorb onto the surface of the platinum catalyst and therefore hampers the reaction kinetics. Additionally, the binder material plays a key role in regulating the hydrophobicity and hydrophilicity of the catalyst layer. Subsequently, the binder controls the electrode–membrane interface that establishes the triple phase boundary between proton conductive electrolyte, electron conductive catalyst, and reactant gases. Moreover, the elevated operating temperatures promote carbon corrosion and therefore degrade the integrity of the catalyst support. These are only some examples how materials properties affect the stability and performance of HT-PEMFCs. For this reason, materials characterization techniques for HT-PEMFCs, either in situ or ex situ, are highly beneficial. Significant progress has recently been made in this field, which enables us to gain a better understanding of underlying processes occurring during fuel cell operation. Various novel tools for characterizing and diagnosing HT-PEMFCs and key components are presented in this review, including FTIR and Raman spectroscopy, confocal Raman microscopy, synchrotron X-ray imaging, X-ray microtomography, and atomic force microscopy. PMID:25671153

Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

PubMed

Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

2008-12-01

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

PubMed Central

Salamone, Monica; Carfì Pavia, Francesco

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.

PubMed

Vetrivel, Kulandaivelu S; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C; Xu, Huaxi; Thinakaran, Gopal

2004-10-22

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes*

PubMed Central

Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C.; Xu, Huaxi; Thinakaran, Gopal

2005-01-01

Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP. PMID:15322084

Radiographic contrast media alterate the localization of actin/band4.9 in the membrane cytoskeleton of human erythrocytes.

PubMed

Franke, R P; Scharnweber, T; Fuhrmann, R; Mrowietz, C; Wenzel, F; Krüger, A; Jung, F

2014-01-01

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

Process for restoring membrane permeation properties

DOEpatents

Pinnau, Ingo; Toy, Lora G.; Casillas, Carlos G.

1997-05-20

A process for restoring the selectivity of high-flee-volume, glassy polymer membranes for condensable components over less-condensable components or non-condensable components of a gas mixture. The process involves exposing the membrane to suitable sorbent vapor, such as propane or butane, thereby reopening the microvoids that make up the free volume. The selectivity of an aged membrane may be restored to 70-100% of its original value. The selectivity of a membrane which is known to age over time can also be maintained by keeping the membrane in a vapor environment when it is not in use.

Process for restoring membrane permeation properties

DOEpatents

Pinnau, I.; Toy, L.G.; Casillas, C.G.

1997-05-20

A process is described for restoring the selectivity of high-free-volume, glassy polymer membranes for condensable components over less-condensable components or non-condensable components of a gas mixture. The process involves exposing the membrane to suitable sorbent vapor, such as propane or butane, thereby reopening the microvoids that make up the free volume. The selectivity of an aged membrane may be restored to 70--100% of its original value. The selectivity of a membrane which is known to age over time can also be maintained by keeping the membrane in a vapor environment when it is not in use. 8 figs.

Cholesterol in myelin biogenesis and hypomyelinating disorders.

PubMed

Saher, Gesine; Stumpf, Sina Kristin

2015-08-01

The largest pool of free cholesterol in mammals resides in myelin membranes. Myelin facilitates rapid saltatory impulse propagation by electrical insulation of axons. This function is achieved by ensheathing axons with a tightly compacted stack of membranes. Cholesterol influences myelination at many steps, from the differentiation of myelinating glial cells, over the process of myelin membrane biogenesis, to the functionality of mature myelin. Cholesterol emerged as the only integral myelin component that is essential and rate-limiting for the development of myelin in the central and peripheral nervous system. Moreover, disorders that interfere with sterol synthesis or intracellular trafficking of cholesterol and other lipids cause hypomyelination and neurodegeneration. This review summarizes recent results on the roles of cholesterol in CNS myelin biogenesis in normal development and under different pathological conditions. This article is part of a Special Issue entitled Brain Lipids. Copyright © 2015 Elsevier B.V. All rights reserved.

Bactericidal Effects and Mechanism of Action of Olanexidine Gluconate, a New Antiseptic

PubMed Central

Iwata, Koushi; Nii, Takuya; Nakata, Hikaru; Tsubotani, Yoshie; Inoue, Yasuhide

2015-01-01

Olanexidine gluconate [1-(3,4-dichlorobenzyl)-5-octylbiguanide gluconate] (development code OPB-2045G) is a new monobiguanide compound with bactericidal activity. In this study, we assessed its spectrum of bactericidal activity and mechanism of action. The minimal bactericidal concentrations of the compound for 30-, 60-, and 180-s exposures were determined with the microdilution method using a neutralizer against 320 bacterial strains from culture collections and clinical isolates. Based on the results, the estimated bactericidal olanexidine concentrations with 180-s exposures were 869 μg/ml for Gram-positive cocci (155 strains), 109 μg/ml for Gram-positive bacilli (29 strains), and 434 μg/ml for Gram-negative bacteria (136 strains). Olanexidine was active against a wide range of bacteria, especially Gram-positive cocci, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and had a spectrum of bactericidal activity comparable to that of commercial antiseptics, such as chlorhexidine and povidone-iodine. In vitro experiments exploring its mechanism of action indicated that olanexidine (i) interacts with the bacterial surface molecules, such as lipopolysaccharide and lipoteichoic acid, (ii) disrupts the cell membranes of liposomes, which are artificial bacterial membrane models, (iii) enhances the membrane permeability of Escherichia coli, (iv) disrupts the membrane integrity of S. aureus, and (v) denatures proteins at relatively high concentrations (≥160 μg/ml). These results indicate that olanexidine probably binds to the cell membrane, disrupts membrane integrity, and its bacteriostatic and bactericidal effects are caused by irreversible leakage of intracellular components. At relatively high concentrations, olanexidine aggregates cells by denaturing proteins. This mechanism differs slightly from that of a similar biguanide compound, chlorhexidine. PMID:25987609

[The effect of dexamethoxin on the integrity of cytoplasmic membrane in gram-positive and gram-negative microorganisms].

PubMed

Shchetina, V N; Belanov, E F; Starobinets, Z G; Volianskiĭ, Iu L

1990-01-01

Decamethoxin is shown to be able to increase membrane permeability of Pseudomonas aeruginosa, Escherichia coli and Micrococcus lysodeikticus, that is confirmed by a loss of compounds with the absorption maximum at 260 nm by cells. Parallel with this the number of viable individuals has fallen and activity of dehydrogenases has been inhibited. The aspartate and alanine aminotransferase activity was not inhibited by decamethoxin and even increased. Decamethoxin lysed the protoplasts of the tested microorganisms. At high decamethoxin concentrations (over 500 micrograms/ml for P. aeruginosa and over 200 mu/ml--for E. coli) the outflow of components from the cells of gram-negative bacteria ceased, that may be associated with the coagulation changes in the cytoplasm. A loss of the low-molecular components by M. lysodeikticus cells and lysis of protoplasts proceeded less intensely than the same processes in the gram-negative microorganisms, that is explained by a less resistance of M. lysodeikticus to decamethoxin and earlier coagulation of the cytoplasm preventing lysis.

3D multifunctional integumentary membranes for spatiotemporal cardiac measurements and stimulation across the entire epicardium

NASA Astrophysics Data System (ADS)

Xu, Lizhi; Gutbrod, Sarah R.; Bonifas, Andrew P.; Su, Yewang; Sulkin, Matthew S.; Lu, Nanshu; Chung, Hyun-Joong; Jang, Kyung-In; Liu, Zhuangjian; Ying, Ming; Lu, Chi; Webb, R. Chad; Kim, Jong-Seon; Laughner, Jacob I.; Cheng, Huanyu; Liu, Yuhao; Ameen, Abid; Jeong, Jae-Woong; Kim, Gwang-Tae; Huang, Yonggang; Efimov, Igor R.; Rogers, John A.

2014-02-01

Means for high-density multiparametric physiological mapping and stimulation are critically important in both basic and clinical cardiology. Current conformal electronic systems are essentially 2D sheets, which cannot cover the full epicardial surface or maintain reliable contact for chronic use without sutures or adhesives. Here we create 3D elastic membranes shaped precisely to match the epicardium of the heart via the use of 3D printing, as a platform for deformable arrays of multifunctional sensors, electronic and optoelectronic components. Such integumentary devices completely envelop the heart, in a form-fitting manner, and possess inherent elasticity, providing a mechanically stable biotic/abiotic interface during normal cardiac cycles. Component examples range from actuators for electrical, thermal and optical stimulation, to sensors for pH, temperature and mechanical strain. The semiconductor materials include silicon, gallium arsenide and gallium nitride, co-integrated with metals, metal oxides and polymers, to provide these and other operational capabilities. Ex vivo physiological experiments demonstrate various functions and methodological possibilities for cardiac research and therapy.

Immunological properties of Micrococcus lysodeikticus membranes.

PubMed

Fukui, Y; Nachbar, M S; Salton, M R

1971-01-01

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.

Uptake of raft components into amyloid β-peptide aggregates and membrane damage.

PubMed

Sasahara, Kenji; Morigaki, Kenichi; Mori, Yasuko

2015-07-15

Amyloid aggregation and deposition of amyloid β-peptide (Aβ) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aβ with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aβ deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aβ in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aβ, uptake of the raft components into Aβ aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aβ deposits induces macroscopic heterogeneity of the membrane structure. Copyright © 2015 Elsevier Inc. All rights reserved.

Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing.

PubMed

Xue, Rui; Liu, Yalong; Zhang, Qingsong; Liang, Congcong; Qin, Huazhen; Liu, Pengfei; Wang, Ke; Zhang, Xiaoyong; Chen, Li; Wei, Yen

2016-08-01

To verify the interaction mechanism between sericin and Escherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin against E. coli as a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin on E. coli and the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for an in vivo study of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing of E. coli cells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. When E. coli cells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction of E. coli Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels. The specific relationship and interaction mechanism between sericin and E. coli cells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing of E. coli cells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequent in vivo results demonstrate that the sericin-poly(N-isopropylacrylamide-N,N'-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Integrated Microfluidic Membrane Transistor Utilizing Chemical Information for On-Chip Flow Control.

PubMed

Frank, Philipp; Schreiter, Joerg; Haefner, Sebastian; Paschew, Georgi; Voigt, Andreas; Richter, Andreas

2016-01-01

Microfluidics is a great enabling technology for biology, biotechnology, chemistry and general life sciences. Despite many promising predictions of its progress, microfluidics has not reached its full potential yet. To unleash this potential, we propose the use of intrinsically active hydrogels, which work as sensors and actuators at the same time, in microfluidic channel networks. These materials transfer a chemical input signal such as a substance concentration into a mechanical output. This way chemical information is processed and analyzed on the spot without the need for an external control unit. Inspired by the development electronics, our approach focuses on the development of single transistor-like components, which have the potential to be used in an integrated circuit technology. Here, we present membrane isolated chemical volume phase transition transistor (MIS-CVPT). The device is characterized in terms of the flow rate from source to drain, depending on the chemical concentration in the control channel, the source-drain pressure drop and the operating temperature.

Integrated Microfluidic Membrane Transistor Utilizing Chemical Information for On-Chip Flow Control

PubMed Central

Frank, Philipp; Schreiter, Joerg; Haefner, Sebastian; Paschew, Georgi; Voigt, Andreas; Richter, Andreas

2016-01-01

Microfluidics is a great enabling technology for biology, biotechnology, chemistry and general life sciences. Despite many promising predictions of its progress, microfluidics has not reached its full potential yet. To unleash this potential, we propose the use of intrinsically active hydrogels, which work as sensors and actuators at the same time, in microfluidic channel networks. These materials transfer a chemical input signal such as a substance concentration into a mechanical output. This way chemical information is processed and analyzed on the spot without the need for an external control unit. Inspired by the development electronics, our approach focuses on the development of single transistor-like components, which have the potential to be used in an integrated circuit technology. Here, we present membrane isolated chemical volume phase transition transistor (MIS-CVPT). The device is characterized in terms of the flow rate from source to drain, depending on the chemical concentration in the control channel, the source-drain pressure drop and the operating temperature. PMID:27571209

Light-modulating pressure sensor with integrated flexible organic light-emitting diode.

PubMed

Cheneler, D; Vervaeke, M; Thienpont, H

2014-05-01

Organic light-emitting diodes (OLEDs) are used almost exclusively for display purposes. Even when implemented as a sensing component, it is rarely in a manner that exploits the possible compliance of the OLED. Here it is shown that OLEDs can be integrated into compliant mechanical micro-devices making a new range of applications possible. A light-modulating pressure sensor is considered, whereby the OLED is integrated with a silicon membrane. It is shown that such devices have potential and advantages over current measurement techniques. An analytical model has been developed that calculates the response of the device. Ray tracing numerical simulations verify the theory and show that the design can be optimized to maximize the resolution of the sensor.

Thinning of PLZT ceramic wafers for sensor integration

NASA Astrophysics Data System (ADS)

Jin, Na; Liu, Weiguo

2010-08-01

Characteristics of transparent PLZT ceramics can be tailored by controlling the component of them, and therefore showed excellent dielectric, piezoelectric, pyroelectric and ferroelectric properties. To integrate the ceramics with microelectronic circuit to realize integrated applications, the ceramic wafers have to be thinned down to micrometer scale in thickness. A7/65/35 PLZT ceramic wafer was selected in this study for the thinning process. Size of the wafer was 10×10mm with an initial thickness of 300μm. A novel membrane transfer process (MTP) was developed for the thinning and integration of the ceramic wafers. In the MTP process, the ceramic wafer was bonded to silicon wafer using a polymer bonding method. Mechanical grinding method was applied to reduce the thickness of the ceramic. To minimize the surface damage in the ceramic wafer caused by the mechanical grinding, magnetorheological finishing (MRF) method was utilized to polish the wafer. White light interference (WLI) apparatus was used to monitor the surface qualities of the grinded and ploished ceramic wafers. For the PLZT membrane obtained from the MTP process, the final thickness of the thinned and polished wafer was 10μm, the surface roughness was below 1nm in rms, and the flatness was better than λ/5.

Dynamics of the Glycophorin A Dimer in Membranes of Native-Like Composition Uncovered by Coarse-Grained Molecular Dynamics Simulations.

PubMed

Flinner, Nadine; Schleiff, Enrico

2015-01-01

Membranes are central for cells as borders to the environment or intracellular organelle definition. They are composed of and harbor different molecules like various lipid species and sterols, and they are generally crowded with proteins. The membrane system is very dynamic and components show lateral, rotational and translational diffusion. The consequence of the latter is that phase separation can occur in membranes in vivo and in vitro. It was documented that molecular dynamics simulations of an idealized plasma membrane model result in formation of membrane areas where either saturated lipids and cholesterol (liquid-ordered character, Lo) or unsaturated lipids (liquid-disordered character, Ld) were enriched. Furthermore, current discussions favor the idea that proteins are sorted into the liquid-disordered phase of model membranes, but experimental support for the behavior of isolated proteins in native membranes is sparse. To gain insight into the protein behavior we built a model of the red blood cell membrane with integrated glycophorin A dimer. The sorting and the dynamics of the dimer were subsequently explored by coarse-grained molecular dynamics simulations. In addition, we inspected the impact of lipid head groups and the presence of cholesterol within the membrane on the dynamics of the dimer within the membrane. We observed that cholesterol is important for the formation of membrane areas with Lo and Ld character. Moreover, it is an important factor for the reproduction of the dynamic behavior of the protein found in its native environment. The protein dimer was exclusively sorted into the domain of Ld character in the model red blood cell plasma membrane. Therefore, we present structural information on the glycophorin A dimer distribution in the plasma membrane in the absence of other factors like e.g. lipid anchors in a coarse grain resolution.

Fourier Transform Infrared and Resonance Raman Spectroscopic Studies of Bacteriorhodopsin.

NASA Astrophysics Data System (ADS)

Earnest, Thomas Nixon

Fourier transform infrared and resonance Raman spectroscopy were used to investigate the structure and function of the light-activated, transmembrane proton pump, bacteriorhodopsin, from the purple membrane of Halobacterium halobium. Bacteriorhodopsin (bR) is a 27,000 dalton integral membrane protein consisting of 248 amino acids with a retinylidene chromophore. Absorption of a photon leads to the translocation of one or two protons from the inside of the cell to the outside. Resonance Raman spectroscopy allows for the study of the configuration of retinal in bR and its photointermediates by the selective enhancement of vibrational modes of the chromophore. This technique was used to determine that the chromophore is attached to lysine-216 in both the bR _{570} and the M _{412} intermediates. In bR with tyrosine-64 selectively nitrated or aminated, the chromophore appears to have the same configuration in that bR _{570} (all- trans) and M _{412} (13- cis) states as it does in unmodified bR. Polarized Fourier transform infrared spectroscopy (FTIR) permits the study of the direction of transition dipole moments arising from molecular vibrations of the protein and the retinal chromophore. The orientation of alpha helical and beta sheet components was determined for bR with the average helical tilt found to lie mostly parallel to the membrane normal. The beta sheet structures also exhibit an IR linear dichroism for the amide I and amide II bands which suggest that the peptide backbone is mostly perpendicular to the membrane plane although it is difficult to determine whether the bands originate from sheet or turn components. The orientation of secondary structure components of the C-1 (residues 72-248) and C-2 (residues 1-71) fragments were also investigated to determine the structure of these putative membrane protein folding intermediates. Polarized, low temperature FTIR -difference spectroscopy was then used to investigate the structure of bR as it undergoes phototransitions from the light-adapted state, bR_{570} , to the K_{630} and M_{412} intermediates. The linear dichroism of C=C and C-C stretching modes, and the hydrogen out-of-plane (HOOP) modes of the chromophore show that the long axis of the polyene is 20-25^ circ out of the membrane plane and that the polyene plane is oriented mostly perpendicular to the membrane plane. Transition dipole moments from protein components are also investigated to determine the orientation of protein groups which undergo changes during the photocycle.

Treatment of mcf-7 breast cancer cells with a red grape wine polyphenol fraction results in disruption of calcium homeostasis and cell cycle arrest causing selective cytotoxicity.

PubMed

Hakimuddin, Fatima; Paliyath, Gopinadhan; Meckling, Kelly

2006-10-04

Food components influence the physiology by modulating gene expression and biochemical pathways within the human body. The disease-preventive roles of several fruit and vegetable components have been related to such properties. Polyphenolic components such as flavonoids are strong antioxidants and induce the expression of several xenobiotic-detoxifying enzymes. The mechanism of selective cytotoxicity induced by red grape wine polyphenols against MCF-7 breast cancer cells was investigated in relation to their interference with calcium homeostasis. MCF-7 cells showed an increase in cytosolic calcium levels within 10 min of treatment with the polyphenols. Immunohistochemical localization of calmodulin with secondary gold-labeled antibodies showed similar levels of gold labeling in both MCF-7 cells and the spontaneously immortalized, normal MCF-10A cell line. MCF-7 cells treated with the red wine polyphenol fraction (RWPF) showed swelling of endoplasmic reticulum, dissolution of the nucleus, and loss of plasma membrane integrity as well as reduced mitochondrial membrane potential. These cells were arrested at the G2/M interphase. By contrast, MCF-10A cells did not show such changes after RWPF treatment. The results suggest that polyphenol-induced calcium release may disrupt mitochondrial function and cause membrane damage, resulting in selective cytotoxicity toward MCF-7 cells. This property could further be developed toward breast cancer prevention strategies either independently or in conjunction with conventional prevention therapies where a positive drug-nutrient interaction can be demonstrated.

Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea.

PubMed

Mulligan, Christopher; Fischer, Marcus; Thomas, Gavin H

2011-01-01

The tripartite ATP-independent periplasmic (TRAP) transporters are the best-studied family of substrate-binding protein (SBP)-dependent secondary transporters and are ubiquitous in prokaryotes, but absent from eukaryotes. They are comprised of an SBP of the DctP or TAXI families and two integral membrane proteins of unequal sizes that form the DctQ and DctM protein families, respectively. The SBP component has a structure comprised of two domains connected by a hinge that closes upon substrate binding. In DctP-TRAP transporters, substrate binding is mediated through a conserved and specific arginine/carboxylate interaction in the SBP. While the SBP component has now been relatively well characterized, the membrane components of TRAP transporters are still poorly understood both in terms of their structure and function. We review the expanding repertoire of substrates and physiological roles for experimentally characterized TRAP transporters in bacteria and discuss mechanistic aspects of these transporters using data primarily from the sialic acid-specific TRAP transporter SiaPQM from Haemophilus influenzae, which suggest that TRAP transporters are high-affinity, Na(+)-dependent unidirectional secondary transporters. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Preparation and Characterization of Graphene Oxide Paper

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

Dikin,D.; Stankovich, S.; Zimney, E.

2007-01-01

Free-standing paper-like or foil-like materials are an integral part of our technological society. Their uses include protective layers, chemical filters, components of electrical batteries or supercapacitors, adhesive layers, electronic or optoelectronic components, and molecular storage. Inorganic 'paper-like' materials based on nanoscale components such as exfoliated vermiculite or mica platelets have been intensively studied and commercialized as protective coatings, high-temperature binders, dielectric barriers and gas-impermeable membranes. Carbon-based flexible graphite foils composed of stacked platelets of expanded graphite have long been used in packing and gasketing applications because of their chemical resistivity against most media, superior sealability over a wide temperature range,more » and impermeability to fluids. The discovery of carbon nanotubes brought about bucky paper, which displays excellent mechanical and electrical properties that make it potentially suitable for fuel cell and structural composite applications. Here we report the preparation and characterization of graphene oxide paper, a free-standing carbon-based membrane material made by flow-directed assembly of individual graphene oxide sheets. This new material outperforms many other paper-like materials in stiffness and strength. Its combination of macroscopic flexibility and stiffness is a result of a unique interlocking-tile arrangement of the nanoscale graphene oxide sheets.« less

Preparation and characterization of graphene oxide paper.

PubMed

Dikin, Dmitriy A; Stankovich, Sasha; Zimney, Eric J; Piner, Richard D; Dommett, Geoffrey H B; Evmenenko, Guennadi; Nguyen, SonBinh T; Ruoff, Rodney S

2007-07-26

Free-standing paper-like or foil-like materials are an integral part of our technological society. Their uses include protective layers, chemical filters, components of electrical batteries or supercapacitors, adhesive layers, electronic or optoelectronic components, and molecular storage. Inorganic 'paper-like' materials based on nanoscale components such as exfoliated vermiculite or mica platelets have been intensively studied and commercialized as protective coatings, high-temperature binders, dielectric barriers and gas-impermeable membranes. Carbon-based flexible graphite foils composed of stacked platelets of expanded graphite have long been used in packing and gasketing applications because of their chemical resistivity against most media, superior sealability over a wide temperature range, and impermeability to fluids. The discovery of carbon nanotubes brought about bucky paper, which displays excellent mechanical and electrical properties that make it potentially suitable for fuel cell and structural composite applications. Here we report the preparation and characterization of graphene oxide paper, a free-standing carbon-based membrane material made by flow-directed assembly of individual graphene oxide sheets. This new material outperforms many other paper-like materials in stiffness and strength. Its combination of macroscopic flexibility and stiffness is a result of a unique interlocking-tile arrangement of the nanoscale graphene oxide sheets.

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Impact evaluation of α-lipoic acid in gamma-irradiated erythrocytes

NASA Astrophysics Data System (ADS)

Desouky, Omar S.; Selim, Nabila S.; Elbakrawy, Eman M.; Rezk, Rezk A.

2011-03-01

This work is intended to study in vitro the ability of lipoic acid to protect erythrocytes against the oxidative damage resulting from exposure to gamma radiation through measurement of their rheological properties and to study the effects of detergent on their membrane solubility and permeability. Different doses of gamma radiation were applied: the most recommended and applied dose (25 Gy), and two higher doses, namely 50 and 100 Gy. The effect of addition of lipoic acid as well as its effect as a radioprotector was tested. The obtained results show changes in structural integrity of the erythrocyte cell membrane components as a result of oxidative damage due to gamma radiation that could be improved by pre-treatment with the antioxidant lipoic acid.

Caveolae.

PubMed

Parton, Robert G; Tillu, Vikas A; Collins, Brett M

2018-04-23

Caveolae are one of the most abundant and striking features of the plasma membrane of many mammalian cell types. These surface pits have fascinated biologists since their discovery by the pioneers of electron microscopy in the middle of the last century, but we are only just starting to understand their multiple functions. Molecular understanding of caveolar formation is advancing rapidly and we now know that sculpting the membrane to generate the characteristic bulb-shaped caveolar pit involves the coordinated action of integral membrane proteins and peripheral membrane coat proteins in a process dependent on their multiple interactions with membrane lipids. The resulting structure is further stabilised by protein complexes at the caveolar neck. Caveolae can bud to generate an endocytic carrier but can also be disassembled in response to specific stimuli to function as a mechanoprotective device. These structures have also been linked to numerous signalling pathways. Here, we will briefly summarise the current molecular and structural understanding of caveolar formation and dynamics, discuss how the crucial structural components of caveolae work together to generate a dynamic sensing domain, and discuss the implications of recent studies on the diverse roles proposed for caveolae in different cells and tissues. Copyright © 2018 Elsevier Ltd. All rights reserved.

Bio-Mimetic Sensors Based on Molecularly Imprinted Membranes

PubMed Central

Algieri, Catia; Drioli, Enrico; Guzzo, Laura; Donato, Laura

2014-01-01

An important challenge for scientific research is the production of artificial systems able to mimic the recognition mechanisms occurring at the molecular level in living systems. A valid contribution in this direction resulted from the development of molecular imprinting. By means of this technology, selective molecular recognition sites are introduced in a polymer, thus conferring it bio-mimetic properties. The potential applications of these systems include affinity separations, medical diagnostics, drug delivery, catalysis, etc. Recently, bio-sensing systems using molecularly imprinted membranes, a special form of imprinted polymers, have received the attention of scientists in various fields. In these systems imprinted membranes are used as bio-mimetic recognition elements which are integrated with a transducer component. The direct and rapid determination of an interaction between the recognition element and the target analyte (template) was an encouraging factor for the development of such systems as alternatives to traditional bio-assay methods. Due to their high stability, sensitivity and specificity, bio-mimetic sensors-based membranes are used for environmental, food, and clinical uses. This review deals with the development of molecularly imprinted polymers and their different preparation methods. Referring to the last decades, the application of these membranes as bio-mimetic sensor devices will be also reported. PMID:25196110

Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

PubMed Central

Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

1998-01-01

The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport.

PubMed

Chiu, Chi-Fang; Ghanekar, Yashoda; Frost, Laura; Diao, Aipo; Morrison, Daniel; McKenzie, Eddie; Lowe, Martin

2008-04-09

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.

Influence of different surfactants on the physicochemical properties of elastic liposomes.

PubMed

Barbosa, R M; Severino, P; Preté, P S C; Santana, M H A

2017-05-01

Elastic liposomes are capable to improve drug transport through the skin by acting as penetration enhancers due to the high fluidity and elasticity of the liposome membranes. Therefore, elastic liposomes were prepared and characterized to facilitate the transdermal transport of bioactive molecules. Liposomes consisted of dimyristoylphosphatidylcholine (DMPC) as the structural component, with different surfactants derived from lauric acid as elastic components: C 12 E 5 (polyoxyethylene-5-lauryl ether), PEG4L (polyethyleneglycol-4-lauryl ester), PEG4DL (polyethylene glycol-4-dilauryl ester), PEG8L (polyethylene glycol-8-lauryl ester) and PEG8DL (polyethylene glycol-8-dilauryl ester). The elastic liposomes were characterized in terms of their phospholipid content, mean diameter, size distribution, elasticity and stability during storage, as well as their ability to incorporate surfactant and permeate through 50 nm pore size membranes. The results showed that the phospholipid phase transition temperature, the fluidity of the lipid bilayer resulting from incorporation of the surfactant and the preservation of particle integrity were factors determining the performance of the elastic liposomes in permeating through nanoporous membranes. The best results were obtained using DMPC combined with the surfactants PEG8L or PEG8DL. The findings demonstrate the potential of using elastic liposomes for transdermal administration of drugs.

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

PubMed

Kato, Akira; Nakamura, Korefumi; Kudo, Hisayuki; Tran, Yen Ha; Yamamoto, Yoko; Doi, Hiroyuki; Hirose, Shigehisa

2007-09-01

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.

Near-ambient solid polymer fuel cell

NASA Technical Reports Server (NTRS)

Holleck, G. L.

1993-01-01

Fuel cells are extremely attractive for extraterrestrial and terrestrial applications because of their high energy conversion efficiency without noise or environmental pollution. Among the various fuel cell systems the advanced polymer electrolyte membrane fuel cells based on sulfonated fluoropolymers (e.g., Nafion) are particularly attractive because they are fairly rugged, solid state, quite conductive, of good chemical and thermal stability and show good oxygen reduction kinetics due to the low specific adsorption of the electrolyte on the platinum catalyst. The objective of this program is to develop a solid polymer fuel cell which can efficiently operate at near ambient temperatures without ancillary components for humidification and/or pressurization of the fuel or oxidant gases. During the Phase 1 effort we fabricated novel integral electrode-membrane structures where the dispersed platinum catalyst is precipitated within the Nafion ionomer. This resulted in electrode-membrane units without interfacial barriers permitting unhindered water diffusion from cathode to anode. The integral electrode-membrane structures were tested as fuel cells operating on H2 and O2 or air at 1 to 2 atm and 10 to 50 C without gas humidification. We demonstrated that cells with completely dry membranes could be self started at room temperature and subsequently operated on dry gas for extended time. Typical room temperature low pressure operation with unoptimized electrodes yielded 100 mA/cm(exp 2) at 0.5V and maximum currents over 300 mA/cm(exp 2) with low platinum loadings. Our results clearly demonstrate that operation of proton exchange membrane fuel cells at ambient conditions is feasible. Optimization of the electrode-membrane structure is necessary to assess the full performance potential but we expect significant gains in weight and volume power density for the system. The reduced complexity will make fuel cells also attractive for smaller and portable power supplies and as replacement for batteries.

Evaluation of energy-distribution of a hybrid microbial fuel cell-membrane bioreactor (MFC-MBR) for cost-effective wastewater treatment.

PubMed

Wang, Jie; Bi, Fanghua; Ngo, Huu-Hao; Guo, Wenshan; Jia, Hui; Zhang, Hongwei; Zhang, Xinbo

2016-01-01

A low-cost hybrid system integrating a membrane-less microbial fuel cell (MFC) with an anoxic/oxic membrane bioreactor (MBR) was studied for fouling mitigation. The appended electric field in the MBR was supplied by the MFC with continuous flow. Supernatant from an anaerobic reactor with low dissolved oxygen was used as feed to the MFC in order to enhance its performance compared with that fed with synthetic wastewater. The voltage output of MFC maintained at 0.52±0.02V with 1000Ω resister. The electric field intensity could reach to 0.114Vcm(-1). Compared with the conventional MBR (CMBR), the contents rather than the components of foulants on the cake layer of fouled MFC-MBR system was significantly reduced. Although only 0.5% of the feed COD was translated into electricity and applied to MBR, the hybrid system showed great feasibility without additional consumption but extracting energy from waste water and significantly enhancing the membrane filterability. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antibacterial action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles against Escherichia coli correlated with molecular chain conformation.

PubMed

Wen, Yan; Yao, Fanglian; Sun, Fang; Tan, Zhilei; Tian, Liang; Xie, Lei; Song, Qingchao

2015-03-01

The action mode of quaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shell nanoparticles (CM-HTCC/PAMAM) against Escherichia coli (E. coli) was investigated via a combination of approaches including measurements of cell membrane integrity, outer membrane (OM) and inner membrane (IM) permeability, and scanning electron microscopy (SEM). CM-HTCC/PAMAM dendrimer nanoparticles likely acted in a sequent event-driven mechanism, beginning with the binding of positively charged groups from nanoparticle surface with negative cell surface, thereby causing the disorganization of cell membrane, and subsequent leakage of intracellular components which might ultimately lead to cell death. Moreover, the chain conformation of polymers was taken into account for a better understanding of the antibacterial action mode by means of viscosity and GPC measurements. High utilization ratio of positive charge and large specific surface area generated from a compacted conformation of CM-HTCC/PAMAM, significantly different from the extended conformation of HTCC, were proposed to be involved in the antibacterial action. Copyright © 2014 Elsevier B.V. All rights reserved.

Recovery of process water from spent emulsions generated in copper cable factory.

PubMed

Karakulski, K; Morawski, A W

2011-02-28

Treatment of waste emulsions generated in the cable factory from copper wire drawing was investigated using the integrated membrane processes: ultrafiltration (UF) and nanofiltration (NF). The application of UF tubular membranes (MWCO 100 kDa) resulted in 98% retention of oil and lubricants, whereas the degree of passage of copper ions (the major component of effluents from cable factory) was 99%. The average permeate flux amounted to 45 l/m(2) h for the transmembrane pressure of 3.5 bar during the UF pretreatment of waste emulsions. The Silt Density Index (SDI) values of UF permeates were appropriate for the application of spiral wound membranes in the NF process. The complete removal of oil and lubricants was achieved in NF process and the content of TOC was reduced by more than 90%. The rejection of copper ions in the NF process was 90% and 98% for NF270 and NF90 membranes (FILMTEC), respectively. The quality of NF permeates allows a direct reuse of treated water for the preparation of fresh emulsion. Copyright © 2010 Elsevier B.V. All rights reserved.

Scalable Fabrication of Integrated Nanophotonic Circuits on Arrays of Thin Single Crystal Diamond Membrane Windows.

PubMed

Piracha, Afaq H; Rath, Patrik; Ganesan, Kumaravelu; Kühn, Stefan; Pernice, Wolfram H P; Prawer, Steven

2016-05-11

Diamond has emerged as a promising platform for nanophotonic, optical, and quantum technologies. High-quality, single crystalline substrates of acceptable size are a prerequisite to meet the demanding requirements on low-level impurities and low absorption loss when targeting large photonic circuits. Here, we describe a scalable fabrication method for single crystal diamond membrane windows that achieves three major goals with one fabrication method: providing high quality diamond, as confirmed by Raman spectroscopy; achieving homogeneously thin membranes, enabled by ion implantation; and providing compatibility with established planar fabrication via lithography and vertical etching. On such suspended diamond membranes we demonstrate a suite of photonic components as building blocks for nanophotonic circuits. Monolithic grating couplers are used to efficiently couple light between photonic circuits and optical fibers. In waveguide coupled optical ring resonators, we find loaded quality factors up to 66 000 at a wavelength of 1560 nm, corresponding to propagation loss below 7.2 dB/cm. Our approach holds promise for the scalable implementation of future diamond quantum photonic technologies and all-diamond photonic metrology tools.

Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

PubMed Central

Tsukazaki, Tomoya; Mori, Hiroyuki; Fukai, Shuya; Numata, Tomoyuki; Perederina, Anna; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo; Vassylyev, Dmitry G.; Nureki, Osamu; Ito, Koreaki

2006-01-01

Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P43212. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery. PMID:16582489

Evidence that the C-terminus of Human Presenilin 1 is Located in the Extra-cytoplasmic Space

PubMed Central

James Turner, R.

2005-01-01

The polytopic membrane protein presenilin 1 (PS1) is a component of the γ-secretase complex that is responsible for the intramembranous cleavage of a number of type I transmembrane proteins including the β-amyloid precursor protein (APP). Mutations of PS1, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer's disease. PS1 contains ten hydrophobic regions (HRs) sufficiently long to be α-helical membrane spanning segments. Most topology models for PS1 place its C-terminal ∼40 amino acids, which include the 10th HR, in the cytosolic space. However, several recent observations suggest that HR 10 may be integrated into the membrane and involved in the interaction between PS1 and APP. We have applied three independent methodologies to investigate the location of HR 10 and the extreme C-terminus of PS1. The results from these methods indicate that HR 10 spans the membrane and that the C-terminal amino acids of PS1 lie in the extra-cytoplasmic space. PMID:15843437

Plastoglobules in algae: A comprehensive comparative study of the presence of major structural and functional components in complex plastids.

PubMed

Lohscheider, Jens N; Río Bártulos, Carolina

2016-08-01

Plastoglobules (PG) are lipophilic droplets attached to thylakoid membranes in higher plants and green algae and are implicated in prenyl lipid biosynthesis. They might also represent a central hub for integration of plastid signals under stress and therefore the adaptation of the thylakoid membrane under such conditions. In Arabidopsis thaliana, PG contain around 30 specific proteins of which Fibrillins (FBN) and Activity of bc1 complex kinases (ABC1K) represent the majority with respect to both number and protein mass. However, nothing is known about the presence of PG in most algal species, which are responsible for about 50% of global primary production. Therefore, we searched the genomes of publicly available algal genomes for components of PG and the associated functional network in order to predict their presence and potential evolutionary conservation of physiological functions. We could identify homologous sequences for core components of PG, like FBN and ABC1K, in most investigated algal species. Furthermore, proteins at central and interesting positions within the PG functional coexpression network were identified. Phylogenetic sequence analysis revealed diversity within FBN and ABC1K sequences among algal species with complex plastids of the red lineage and large differences compared with green lineage species. Two types of FBN were detected that differ in their isoelectric point which seems to correlate with subcellular localization. Subgroups of FBN were shared between many investigated species and modeling of their 3D-structure implied a conserved structure. FBN and ABC1K are essential structural and functional components of PG. Their occurrence in investigated algal species suggests presence of PG therein and functions in prenyl lipid metabolism and adaptation of the thylakoid membrane that are conserved during evolution. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantifying Square Membrane Wrinkle Behavior Using MITC Shell Elements

NASA Technical Reports Server (NTRS)

Jacobson, Mindy B.; Iwasa, Takashi; Natori, M. C.

2004-01-01

For future membrane based structures, quantified predictions of membrane wrinkling behavior in terms of amplitude, angle and wavelength are needed to optimize the efficiency and integrity of such structures, as well as their associated control systems. For numerical analyses performed in the past, limitations on the accuracy of membrane distortion simulations have often been related to the assumptions made while using finite elements. Specifically, this work demonstrates that critical assumptions include: effects of gravity. supposed initial or boundary conditions, and the type of element used to model the membrane. In this work, a 0.2 square meter membrane is treated as a structural material with non-negligible bending stiffness. Mixed Interpolation of Tensorial Components (MTTC) shell elements are used to simulate wrinkling behavior due to a constant applied in-plane shear load. Membrane thickness, gravity effects, and initial imperfections with respect to flatness were varied in numerous nonlinear analysis cases. Significant findings include notable variations in wrinkle modes for thickness in the range of 50 microns to 1000 microns, which also depend on the presence of an applied gravity field. However, it is revealed that relationships between overall strain energy density for cases with differing initial conditions are independent of assumed initial con&tions. In addition, analysis results indicate that the relationship between amplitude scale (W/t) and structural scale (L/t) is linear in the presence of a gravity field.

Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

DOE PAGES

Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; ...

2015-12-22

Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

An approach to industrial water conservation--a case study involving two large manufacturing companies based in Australia.

PubMed

Agana, Bernard A; Reeve, Darrell; Orbell, John D

2013-01-15

This study presents the application of an integrated water management strategy at two large Australian manufacturing companies that are contrasting in terms of their respective products. The integrated strategy, consisting of water audit, pinch analysis and membrane process application, was deployed in series to systematically identify water conservation opportunities. Initially, a water audit was deployed to completely characterize all water streams found at each production site. This led to the development of a water balance diagram which, together with water test results, served as a basis for subsequent enquiry. After the water audit, commercially available water pinch software was utilized to identify possible water reuse opportunities, some of which were subsequently implemented on site. Finally, utilizing a laboratory-scale test rig, membrane processes such as UF, NF and RO were evaluated for their suitability to treat the various wastewater streams. The membranes tested generally showed good contaminant rejection rates, slow flux decline rates, low energy usage and were well suited for treatment of specific wastewater streams. The synergy between the various components of this strategy has the potential to reduce substantial amounts of Citywater consumption and wastewater discharge across a diverse range of large manufacturing companies. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

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

Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.

Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

Two-component fluid membranes near repulsive walls: Linearized hydrodynamics of equilibrium and nonequilibrium states.

PubMed

Sankararaman, Sumithra; Menon, Gautam I; Sunil Kumar, P B

2002-09-01

We study the linearized hydrodynamics of a two-component fluid membrane near a repulsive wall, using a model that incorporates curvature-concentration coupling as well as hydrodynamic interactions. This model is a simplified version of a recently proposed one [J.-B. Manneville et al., Phys. Rev. E 64, 021908 (2001)] for nonequilibrium force centers embedded in fluid membranes, such as light-activated bacteriorhodopsin pumps incorporated in phospholipid egg phosphatidyl choline (EPC) bilayers. The pump-membrane system is modeled as an impermeable, two-component bilayer fluid membrane in the presence of an ambient solvent, in which one component, representing active pumps, is described in terms of force dipoles displaced with respect to the bilayer midpoint. We first discuss the case in which such pumps are rendered inactive, computing the mode structure in the bulk as well as the modification of hydrodynamic properties by the presence of a nearby wall. These results should apply, more generally, to equilibrium fluid membranes comprised of two components, in which the effects of curvature-concentration coupling are significant, above the threshold for phase separation. We then discuss the fluctuations and mode structure in the steady state of active two-component membranes near a repulsive wall. We find that proximity to the wall smoothens membrane height fluctuations in the stable regime, resulting in a logarithmic scaling of the roughness even for initially tensionless membranes. This explicitly nonequilibrium result is a consequence of the incorporation of curvature-concentration coupling in our hydrodynamic treatment. This result also indicates that earlier scaling arguments which obtained an increase in the roughness of active membranes near repulsive walls upon neglecting the role played by such couplings may need to be reevaluated.

Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

PubMed

Yang, Yoosoo; Hong, Yeonsun; Nam, Gi-Hoon; Chung, Jin Hwa; Koh, Eunee; Kim, In-San

2017-04-01

An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diffusion of molecules and macromolecules in thylakoid membranes.

PubMed

Kirchhoff, Helmut

2014-04-01

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components. Copyright © 2013 Elsevier B.V. All rights reserved.

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

PubMed Central

Miller, Thomas F.

2017-01-01

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency. PMID:28328943

A membrane stirrer for product recovery and substrate feeding.

PubMed

Femmer, T; Carstensen, F; Wessling, M

2015-02-01

During fermentation processes, in situ product recovery (ISPR) using submerged membranes allows a continuous operation mode with effective product removal. Continuous recovery reduces product inhibition and organisms in the reactor are not exposed to changing reaction conditions. For an effective in situ product removal, submerged membrane systems should have a sufficient large membrane area and an anti-fouling concept integrated in a compact device for the limited space in a lab-scale bioreactor. We present a new membrane stirrer with integrated filtration membranes on the impeller blades as well as an integrated gassing concept in an all-in-one device. The stirrer is fabricated by rapid prototyping and is equipped with a commercial micromesh membrane. Filtration performance is tested using a yeast cell suspension with different stirring speeds and aeration fluxes. We reduce membrane fouling by backflushing through the membrane with the product stream. © 2014 Wiley Periodicals, Inc.

Integrable structure in discrete shell membrane theory

PubMed Central

Schief, W. K.

2014-01-01

We present natural discrete analogues of two integrable classes of shell membranes. By construction, these discrete shell membranes are in equilibrium with respect to suitably chosen internal stresses and external forces. The integrability of the underlying equilibrium equations is proved by relating the geometry of the discrete shell membranes to discrete O surface theory. We establish connections with generalized barycentric coordinates and nine-point centres and identify a discrete version of the classical Gauss equation of surface theory. PMID:24808755

Integrable structure in discrete shell membrane theory.

PubMed

Schief, W K

2014-05-08

We present natural discrete analogues of two integrable classes of shell membranes. By construction, these discrete shell membranes are in equilibrium with respect to suitably chosen internal stresses and external forces. The integrability of the underlying equilibrium equations is proved by relating the geometry of the discrete shell membranes to discrete O surface theory. We establish connections with generalized barycentric coordinates and nine-point centres and identify a discrete version of the classical Gauss equation of surface theory.

Membrane Protein Incorporation into Nano-Bioelectronics: An insight into Rhodopsin Controlled SiNW-FET Devices

NASA Astrophysics Data System (ADS)

Tunuguntla, Ramya

Biological systems use different energy sources to interact with their environments by creating ion gradients, membrane electric potentials, or a proton motive force to accomplish strikingly complex tasks on the nanometer length scale, such as energy harvesting, and whole organism replication. Most of this activity involves a vast arsenal of active and passive ion channels, membrane receptors and ion pumps that mediate complex and precise transport across biological membranes. Despite the remarkable rate of progress exhibited by modern microelectronic devices, they still cannot compete with the efficiency and precision of biological systems on the component level. At the same time, the sophistication of these molecular machines provides an excellent opportunity to use them in hybrid bioelectronic devices where such a combination could deliver enhanced electronic functionality and enable seamless bi-directional interfaces between man-made and biological assemblies. Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex-vivo devices such as electronic biosensors, thin-film protein arrays, or bio-fuel cells. Since most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In our work, we have explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes. One-dimensional inorganic nanostructures, which have critical dimensions comparable to the sizes of biological molecules, form an excellent materials platform for building such integrated structures. Researchers already use silicon nanowire-based field effect transistors functionalized with molecular recognition sites in a diverse array of biosensors. In our group, we have been developing a platform for integration of membrane protein functionality and electronic devices using a 1-D phospholipid bilayer device architecture. In these devices, the membrane proteins reside within the lipid bilayer that covers a nanowire channel of a field-effect transistor. This lipid bilayer performs several functions: it shields the nanowire from the solution species; it serves as a native-like environment for membrane proteins and preserves their functionality, integrity, and even vectorality. In this work, we show that a 1-D bilayer device incorporating a rhodopsin proton pump allows us to couple light-driven proton transport to a bioelectronic circuit. We also report that we were able to adapt another distinctive feature of biological signal processing---their widespread use of modifiers, co-factors, and mediator molecules---to regulate and fine-tune the operational characteristics of the bioelectronic device. In our example, we use co-assembly of protein channels and ionophores in the 1-D bilayer to modify the device output levels and response time.

Application of Novel Anion-Exchange Blend Membranes (AEBMs) to Vanadium Redox Flow Batteries.

PubMed

Cho, Hyeongrae; Krieg, Henning M; Kerres, Jochen A

2018-06-19

Both cation-exchange membranes and anion-exchange membranes are used as ion conducting membranes in vanadium redox flow batteries (VRFBs). Anion-exchange membranes (AEMs) are applied in vanadium redox flow batteries due to the high blocking property of vanadium ions via the Donnan exclusion effect. In this study, novel anion-exchange blend membranes (AEBMs) were prepared, characterized, and applied in VRFBs. Bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide), poly[(1-(4,4′-diphenylether)-5-oxybenzimidazole)-benzimidazole] (PBI-OO) and sulfonated polyether sulfone polymer were combined to prepare 3-component AEBMs with 1,2,4,5-tetramethylimidazole (TMIm) for quaternization. 3-component AEBMs showed significantly enhanced chemical and mechanical properties compared with those of 2-component AEBMs, resulting in an improved performance in VRFBs. The compositions of the anion-exchange polymers in 3-component AEBMs were systematically varied to optimize the AEBMs for the redox-flow battery application. While the 3-component AEBMs showed comparable efficiencies with Nafion ® 212 membranes, they displayed improved vanadium ions cross-over as was confirmed by open circuit voltage tests and capacity fade tests conducted in VRFBs. In addition, one of the synthesized 3-component AEBM had a superior coulombic efficiency and capacity retention in a charging⁻discharging test over 300 cycles at a current density of 40 mA/cm². It can thus be concluded that 3-component AEBMs are promising candidates for long-term operation in VRFBs.

Membrane permeation process for dehydration of organic liquid mixtures using sulfonated ion-exchange polyalkene membranes

DOEpatents

Cabasso, Israel; Korngold, Emmanuel

1988-01-01

A membrane permeation process for dehydrating a mixture of organic liquids, such as alcohols or close boiling, heat sensitive mixtures. The process comprises causing a component of the mixture to selectively sorb into one side of sulfonated ion-exchange polyalkene (e.g., polyethylene) membranes and selectively diffuse or flow therethrough, and then desorbing the component into a gas or liquid phase on the other side of the membranes.

Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant.

PubMed

Turner, Lorinda; Praszkier, Judyta; Hutton, Melanie L; Steer, David; Ramm, Georg; Kaparakis-Liaskos, Maria; Ferrero, Richard L

2015-08-01

Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes. © 2015 John Wiley & Sons Ltd.

Membrane-augmented cryogenic methane/nitrogen separation

DOEpatents

Lokhandwala, Kaaeid

1997-01-01

A membrane separation process combined with a cryogenic separation process for treating a gas stream containing methane, nitrogen and at least one other component. The membrane separation process works by preferentially permeating methane and the other component and rejecting nitrogen. The process is particularly useful in removing components such as water, carbon dioxide or C.sub.3+ hydrocarbons that might otherwise freeze and plug the cryogenic equipment.

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

PubMed Central

Casey, Amanda K.; Chen, Shuliang; Novick, Peter; Ferro-Novick, Susan; Wente, Susan R.

2015-01-01

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions. PMID:26041935

Effects of Bloom-Forming Algae on Fouling of Integrated Membrane Systems in Seawater Desalination

ERIC Educational Resources Information Center

Ladner, David Allen

2009-01-01

Combining low- and high-pressure membranes into an integrated membrane system is an effective treatment strategy for seawater desalination. Low-pressure microfiltration (MF) and ultrafiltration (UF) membranes remove particulate material, colloids, and high-molecular-weight organics leaving a relatively foulant-free salt solution for treatment by…

Solid-state membrane module

DOEpatents

Gordon, John Howard [Salt Lake City, UT; Taylor, Dale M [Murray, UT

2011-06-07

Solid-state membrane modules comprising at least one membrane unit, where the membrane unit has a dense mixed conducting oxide layer, and at least one conduit or manifold wherein the conduit or manifold comprises a dense layer and at least one of a porous layer and a slotted layer contiguous with the dense layer. The solid-state membrane modules may be used to carry out a variety of processes including the separating of any ionizable component from a feedstream wherein such ionizable component is capable of being transported through a dense mixed conducting oxide layer of the membrane units making up the membrane modules. For ease of construction, the membrane units may be planar.

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

PubMed Central

Hönlinger, A; Bömer, U; Alconada, A; Eckerskorn, C; Lottspeich, F; Dietmeier, K; Pfanner, N

1996-01-01

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase. Images PMID:8641278

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

PubMed Central

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S.; Stenvang, Marcel; Otzen, Daniel E.

2014-01-01

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. PMID:25304506

The antimicrobial mechanism of action of epsilon-poly-l-lysine.

PubMed

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S; Stenvang, Marcel; Otzen, Daniel E; Meyer, Rikke L

2014-12-01

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

PubMed Central

Qin, Wenxia; Wunderley, Lydia; Barrett, Anne L.; High, Stephen; Woodman, Philip G.

2016-01-01

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins. PMID:27582497

Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins.

PubMed

Maric, Martina; Haugo, Alison C; Dauer, William; Johnson, David; Roller, Richard J

2014-07-01

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Unveiling TRPV1 Spatio-Temporal Organization in Live Cell Membranes

PubMed Central

Storti, Barbara; Di Rienzo, Carmine; Cardarelli, Francesco; Bizzarri, Ranieri; Beltram, Fabio

2015-01-01

Transient Receptor Potential Vanilloid 1 (TRPV1) is a non-selective cation channel that integrates several stimuli into nociception and neurogenic inflammation. Here we investigated the subtle TRPV1 interplay with candidate membrane partners in live cells by a combination of spatio-temporal fluctuation techniques and fluorescence resonance energy transfer (FRET) imaging. We show that TRPV1 is split into three populations with fairly different molecular properties: one binding to caveolin-1 and confined into caveolar structures, one actively guided by microtubules through selective binding, and one which diffuses freely and is not directly implicated in regulating receptor functionality. The emergence of caveolin-1 as a new interactor of TRPV1 evokes caveolar endocytosis as the main desensitization pathway of TRPV1 receptor, while microtubule binding agrees with previous data suggesting the receptor stabilization in functional form by these cytoskeletal components. Our results shed light on the hitherto unknown relationships between spatial organization and TRPV1 function in live-cell membranes. PMID:25764349

Lipid Cell Biology: A Focus on Lipids in Cell Division.

PubMed

Storck, Elisabeth M; Özbalci, Cagakan; Eggert, Ulrike S

2018-06-20

Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

Removal Efficiency of COD, Total P and Total N Components from Municipal Wastewater using Hollow-fibre MBR.

PubMed

Petrinić, Irena; Curlin, Mirjana; Korenak, Jasmina; Simonič, Marjana

2011-06-01

The membrane bioreactor (MBR) integrates well within the conventionally activated sludge system regarding advanced membrane separation for wastewater treatment. Over the last decade, a number of MBR systems have been constructed worldwide and this system is now accepted as a technology of choice for wastewater treatment especially for municipal wastewater. The aim of this work was to investigate and compare submerged MBR with conventionally-activated sludge system for the treatment of municipal wastewater in Maribor, Slovenia. It can be concluded from the results, that the efficiencies being determined by the parameters were satisfied, such as, chemical oxygen demand, total phosphorous, and total nitrogen, which were 97%, 75%, and 90%, respectively. The efficiencies of ultrafiltration membrane for the same parameters were also determined, and compared with biological treatment. The results of this analysis show an additional effect regarding an improvement in the quality of the permeate but primary treatment is also very important. For successfully application of MBR system smaller grid for primary treatment is needed.

Biological accumulation of tellurium nanorod structures via reduction of tellurite by Shewanella oneidensis MR-1.

PubMed

Kim, Dong-Hun; Kanaly, Robert A; Hur, Hor-Gil

2012-12-01

The dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, reduced tellurite (Te(IV), TeO(3)(2-)) to elemental tellurium under anaerobic conditions resulting in the intracellular accumulation of needle shaped crystalline Te(0) nanorods. Fatty acid analyses showed that toxic Te(IV) increased the unsaturated fatty acid composition of the lipid components of the cell membrane, implying a deconstruction of the integrity of the cellular membrane structure. The current results suggest that dissimilatory metal reducing bacteria such as S. oneidensis MR-1 may play an important role in recycling toxic tellurium elements, and may be applied as a novel selective biological filter via the accumulation of industry-applicable rare materials, Te(0) nanorods, in the cell. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis and Characterisation of ETS-10/Acetate-based Ionic Liquid/Chitosan Mixed Matrix Membranes for CO2/N2 Permeation.

PubMed

Casado-Coterillo, Clara; Del Mar López-Guerrero, María; Irabien, Angel

2014-06-19

Mixed matrix membranes (MMMs) were prepared by incorporating organic surfactant-free hydrothermally synthesised ETS-10 and 1-ethyl-3-methylimidazolium acetate ionic liquid (IL) to chitosan (CS) polymer matrix. The membrane material characteristics and permselectivity performance of the two-component membranes were compared with the three-component membrane and the pure CS membrane. The addition of IL increased CO2 solubility of the polymer, and, thus, the CO2 affinity was maintained for the MMMs, which can be correlated with the crystallinity, measured by FT-IR, and void fraction calculations from differences between theoretical and experimental densities. The mechanical resistance was enhanced by the ETS-10 nanoparticles, and flexibility decreased in the two-component ETS-10/CS MMMs, but the flexibility imparted by the IL remained in three-component ETS-10/IL/CS MMMs. The results of this work provide insight into another way of facing the adhesion challenge in MMMs and obtain CO2 selective MMMs from renewable or green chemistry materials.

Indentification and Analysis of Occludin Phosphosites: A Combined Mass Spectroscoy and Bioinformatics Approach

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

Sundstrom, J.; Tash, B; Murakami, T

2009-01-01

The molecular function of occludin, an integral membrane component of tight junctions, remains unclear. VEGF-induced phosphorylation sites were mapped on occludin by combining MS data analysis with bioinformatics. In vivo phosphorylation of Ser490 was validated and protein interaction studies combined with crystal structure analysis suggest that Ser490 phosphorylation attenuates the interaction between occludin and ZO-1. This study demonstrates that combining MS data and bioinformatics can successfully identify novel phosphorylation sites from limiting samples.

Membrane-augmented cryogenic methane/nitrogen separation

DOEpatents

Lokhandwala, K.

1997-07-15

A membrane separation process is described which is combined with a cryogenic separation process for treating a gas stream containing methane, nitrogen and at least one other component. The membrane separation process works by preferentially permeating methane and the other component and rejecting nitrogen. The process is particularly useful in removing components such as water, carbon dioxide or C{sub +2} hydrocarbons that might otherwise freeze and plug the cryogenic equipment. 10 figs.

3D multifunctional integumentary membranes for spatiotemporal cardiac measurements and stimulation across the entire epicardium

PubMed Central

Xu, Lizhi; Gutbrod, Sarah R.; Bonifas, Andrew P.; Su, Yewang; Sulkin, Matthew S.; Lu, Nanshu; Chung, Hyun-Joong; Jang, Kyung-In; Liu, Zhuangjian; Ying, Ming; Lu, Chi; Webb, R. Chad; Kim, Jong-Seon; Laughner, Jacob I.; Cheng, Huanyu; Liu, Yuhao; Ameen, Abid; Jeong, Jae-Woong; Kim, Gwang-Tae; Huang, Yonggang; Efimov, Igor R.; Rogers, John A.

2015-01-01

Means for high-density multiparametric physiological mapping and stimulation are critically important in both basic and clinical cardiology. Current conformal electronic systems are essentially 2D sheets, which cannot cover the full epicardial surface or maintain reliable contact for chronic use without sutures or adhesives. Here we create 3D elastic membranes shaped precisely to match the epicardium of the heart via the use of 3D printing, as a platform for deformable arrays of multifunctional sensors, electronic and optoelectronic components. Such integumentary devices completely envelop the heart, in a form-fitting manner, and possess inherent elasticity, providing a mechanically stable bioti-/abiotic interface during normal cardiac cycles. Component examples range from actuators for electrical, thermal and optical stimulation, to sensors for pH, temperature and mechanical strain. The semiconductor materials include silicon, gallium arsenide and gallium nitride, co-integrated with metals, metal oxides and polymers, to provide these and other operational capabilities. Ex vivo physiological experiments demonstrate various functions and methodological possibilities for cardiac research and therapy. PMID:24569383

Tracking variations in fluorescent-dissolved organic matter in an aerobic submerged membrane bioreactor using excitation-emission matrix spectra combined with parallel factor analysis.

PubMed

Hur, Jin; Shin, Jaewon; Kang, Minsun; Cho, Jinwoo

2014-08-01

In this study, the variations in the fluorescent components of dissolved organic matter (DOM) were tracked for an aerobic submerged membrane bioreactor (MBR) at three different operation stages (cake layer formation, condensation, and after cleaning). The fluorescent DOM was characterized using excitation-emission matrix (EEM) spectroscopy combined with parallel factor analysis (PARAFAC). Non-aromatic carbon structures appear to be actively involved in the membrane fouling for the cake layer formation stage as revealed by much higher UV-absorbing DOM per organic carbon found in the effluent versus those inside the reactor. Four fluorescent components were successfully identified from the reactor and the effluent DOMs by EEM-PARAFAC modeling. Among those in the reactor, microbial humic-like fluorescence was the most abundant component at the cake layer formation stage and tryptophan-like fluorescence at the condensation stage. In contrast to the reactor, relatively similar composition of the PARAFAC components was exhibited for the effluent at all three stages. Tryptophan-like fluorescence displayed the largest difference between the reactor and the effluent, suggesting that this component could be a good tracer for membrane fouling. It appears that the fluorescent DOM was involved in membrane fouling by cake layer formation rather than by internal pore adsorption because its difference between the reactor and the effluent was the highest among all the four components, even after the membrane cleaning. Our study provided an insight into the fate and the behavior fluorescent DOM components for an MBR system, which could be an indicator of the membrane fouling.

Effect of biocompatible polymers on the structural integrity of lipid bilayers under external stimuli

NASA Astrophysics Data System (ADS)

Wang, Jia-Yu; Kausik, Ravinath; Chen, Chi-Yuan; Han, Song-I.; Marks, Jeremy; Lee, Ka Yee

2010-03-01

Cell membrane dysfunction due to loss of structural integrity is the pathology of tissue death in trauma and common diseases. It is now established that certain biocompatible polymers, such as Poloxamer 188, Poloxamine 1107 and polyethylene glycol (PEG), are effective in sealing of injured cell membranes, and able to prevent acute necrosis. Despite these broad applications of these polymers for human health, the fundamental mechanisms by which these polymers interact with cell membranes are still under debate. Here, the effects of a group of biocompatible polymers on phospholipid membrane integrity under osmotic and oxidative stress were explored using giant unilamellar vesicles as model cell membranes. Our results suggest that the adsorption of the polymers on the membrane surface is responsible for the cell membrane resealing process due to its capability of slowing down the surface hydration dynamics.

Efficient ethanol recovery from yeast fermentation broth with integrated distillation-membrane process

EPA Science Inventory

A hybrid process integrating vapor stripping with vapor compression and vapor permeation membrane separation, termed Membrane Assisted Vapor Stripping (MAVS), was evaluated for recovery and dehydration of ethanol from aqueous solution as an alternative to conventional distillatio...

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

PubMed

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane. Copyright © 2017 American Society for Microbiology.

High Pressure Electrolyzer System Evaluation

NASA Technical Reports Server (NTRS)

Prokopius, Kevin; Coloza, Anthony

2010-01-01

This report documents the continuing efforts to evaluate the operational state of a high pressure PEM based electrolyzer located at the NASA Glenn Research Center. This electrolyzer is a prototype system built by General Electric and refurbished by Hamilton Standard (now named Hamilton Sunstrand). It is capable of producing hydrogen and oxygen at an output pressure of 3000 psi. The electrolyzer has been in storage for a number of years. Evaluation and testing was performed to determine the state of the electrolyzer and provide an estimate of the cost for refurbishment. Pressure testing was performed using nitrogen gas through the oxygen ports to ascertain the status of the internal membranes and seals. It was determined that the integrity of the electrolyzer stack was good as there were no appreciable leaks in the membranes or seals within the stack. In addition to the integrity testing, an itemized list and part cost estimate was produced for the components of the electrolyzer system. An evaluation of the system s present state and an estimate of the cost to bring it back to operational status was also produced.

Thin film fabrication and system integration test run for a microactuator for a tuneable lens

NASA Astrophysics Data System (ADS)

Hoheisel, Dominik; Rissing, Lutz

2014-03-01

An electromagnetic microactuator, for controlling of a tuneable lens, with an integrated electrostatic element is fabricated by thin film technology. The actuator consists of two parts: the first part with microcoil and flux guide and the second part with a ring shaped back iron on a polyimide membrane. The back iron is additionally useable as electrode for electrostatic measurement of the air gap and for electrostatic actuation. By attracting the back iron an optical liquid is displaced and forms a liquid lens inside the back iron ring covered by the membrane. For testing the thin film fabrication sequence, up-scaled systems are generated in a test run. To fabricate the flux guide in an easy and quick way, a Ni-Fe foil with a thickness of 50 μm is laminated on the Si-wafer. This foil is also utilized in the following fabrication sequence as seed layer for electroplating. Compared to Ni-Fe structures deposited by electroplating, the foil is featuring better soft magnetic properties. The foil is structured by wet chemical etching and the backside of the wafer is structured by deep reactive ion etching (DRIE). For post fabrication thinning, the polyimide membrane is treated by oxygen plasma etching. To align the back iron to the microcoil and the flux guide, a flip-chip-bonder is used during test run of system integration. To adjust a constant air gap, a water solvable polymer is tested. A two component epoxy and a polyimide based glue are compared for their bonding properties of the actuator parts.

The absence of chlorophyll b affects lateral mobility of photosynthetic complexes and lipids in grana membranes of Arabidopsis and barley chlorina mutants.

PubMed

Tyutereva, Elena V; Evkaikina, Anastasiia I; Ivanova, Alexandra N; Voitsekhovskaja, Olga V

2017-09-01

The lateral mobility of integral components of thylakoid membranes, such as plastoquinone, xanthophylls, and pigment-protein complexes, is critical for the maintenance of efficient light harvesting, high rates of linear electron transport, and successful repair of damaged photosystem II (PSII). The packaging of the photosynthetic pigment-protein complexes in the membrane depends on their size and stereometric parameters which in turn depend on the composition of the complexes. Chlorophyll b (Chlb) is an important regulator of antenna size and composition. In this study, the lateral mobility (the mobile fraction size) of pigment-protein complexes and lipids in grana membranes was analyzed in chlorina mutants of Arabidopsis and barley lacking Chlb. In the Arabidopsis ch1-3 mutant, diffusion of membrane lipids decreased as compared to wild-type plants, but the diffusion of photosynthetic complexes was not affected. In the barley chlorina f2 3613 mutant, the diffusion of pigment-protein complexes significantly decreased, while the diffusion of lipids increased, as compared to wild-type plants. We propose that the size of the mobile fractions of pigment-protein complexes in grana membranes in vivo is higher than reported previously. The data are discussed in the context of the protein composition of antennae, characteristics of the plastoquinone pool, and production of reactive oxygen species in leaves of chlorina mutants.

Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

PubMed

Kessels, Michael M; Qualmann, Britta

2015-09-01

A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions. © 2015. Published by The Company of Biologists Ltd.

A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

PubMed

Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

2014-01-01

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

NASA Technical Reports Server (NTRS)

Miedema, H.; Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1999-01-01

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

PubMed

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

Onion cells after high pressure and thermal processing: comparison of membrane integrity changes using different analytical methods and impact on tissue texture.

PubMed

Gonzalez, Maria E; Anthon, Gordon E; Barrett, Diane M

2010-09-01

Two different analytical methods were evaluated for their capacity to provide quantitative information on onion cell membrane permeability and integrity after high pressure and thermal processing and to study the impact of these processing treatments on cell compartmentalization and texture quality. To determine changes in cell membrane permeability and/or integrity the methodologies utilized were: (1) measurement of a biochemical product, pyruvate, formed as a result of membrane permeabilization followed by enzymatic activity and (2) leakage of electrolytes into solution. These results were compared to previously determined methods that quantified cell viability and ¹H-NMR T(2) of onions. These methods allowed for the monitoring of changes in the plasma and tonoplast membranes after high pressure or thermal processing. High pressure treatments consisted of 5 min holding times at 50, 100, 200, 300, or 600 MPa. Thermal treatments consisted of 30 min water bath exposure to 40, 50, 60, 70, or 90 °C. There was strong agreement between the methods in the determination of the ranges of high pressure and temperature that induce changes in the integrity of the plasma and tonoplast membranes. Membrane rupture could clearly be identified at 300 MPa and above in high pressure treatments and at 60 °C and above in the thermal treatments. Membrane destabilization effects could already be visualized following the 200 MPa and 50 °C treatments. The texture of onions was influenced by the state of the membranes and was abruptly modified once membrane integrity was lost. In this study, we used chemical, biochemical, and histological techniques to obtain information on cell membrane permeability and onion tissue integrity after high pressure and thermal processing. Because there was strong agreement between the various methods used, it is possible to implement something relatively simple, such as ion leakage, into routine quality assurance measurements to determine the severity of preservation methods and the shelf life of processed vegetables.

Dynamics of the Glycophorin A Dimer in Membranes of Native-Like Composition Uncovered by Coarse-Grained Molecular Dynamics Simulations

PubMed Central

Flinner, Nadine; Schleiff, Enrico

2015-01-01

Membranes are central for cells as borders to the environment or intracellular organelle definition. They are composed of and harbor different molecules like various lipid species and sterols, and they are generally crowded with proteins. The membrane system is very dynamic and components show lateral, rotational and translational diffusion. The consequence of the latter is that phase separation can occur in membranes in vivo and in vitro. It was documented that molecular dynamics simulations of an idealized plasma membrane model result in formation of membrane areas where either saturated lipids and cholesterol (liquid-ordered character, Lo) or unsaturated lipids (liquid-disordered character, Ld) were enriched. Furthermore, current discussions favor the idea that proteins are sorted into the liquid-disordered phase of model membranes, but experimental support for the behavior of isolated proteins in native membranes is sparse. To gain insight into the protein behavior we built a model of the red blood cell membrane with integrated glycophorin A dimer. The sorting and the dynamics of the dimer were subsequently explored by coarse-grained molecular dynamics simulations. In addition, we inspected the impact of lipid head groups and the presence of cholesterol within the membrane on the dynamics of the dimer within the membrane. We observed that cholesterol is important for the formation of membrane areas with Lo and Ld character. Moreover, it is an important factor for the reproduction of the dynamic behavior of the protein found in its native environment. The protein dimer was exclusively sorted into the domain of Ld character in the model red blood cell plasma membrane. Therefore, we present structural information on the glycophorin A dimer distribution in the plasma membrane in the absence of other factors like e.g. lipid anchors in a coarse grain resolution. PMID:26222139

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

PubMed Central

Li, H M; Chen, L J

1996-01-01

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

Tracking inorganic foulants irreversibly accumulated on low-pressure membranes for treating surface water.

PubMed

Yamamura, Hiroshi; Kimura, Katsuki; Higuchi, Kumiko; Watanabe, Yoshimasa; Ding, Qing; Hafuka, Akira

2015-12-15

While low-pressure membrane filtration processes (i.e., microfiltration and ultrafiltration) can offer precise filtration than sand filtration, they pose the problem of reduced efficiency due to membrane fouling. Although many studies have examined membrane fouling by organic substances, there is still not enough data available concerning membrane fouling by inorganic substances. The present research investigated changes in the amounts of inorganic components deposited on the surface of membrane filters over time using membrane specimens sampled thirteen times at arbitrary time intervals during pilot testing in order to determine the mechanism by which irreversible fouling by inorganic substances progresses. The experiments showed that the inorganic components that primarily contribute to irreversible fouling vary as filtration continues. It was discovered that, in the initial stage of operation, the main membrane-fouling substance was iron, whereas the primary membrane-fouling substances when operation finished were manganese, calcium, and silica. The amount of iron accumulated on the membrane increased up to the thirtieth day of operation, after which it reached a steady state. After the accumulation of iron became static, subsequent accumulation of manganese was observed. The fact that the removal rates of these inorganic components also increased gradually shows that the size of the exclusion pores of the membrane filter narrows as operation continues. Studying particle size distributions of inorganic components contained in source water revealed that while many iron particles are approximately the same size as membrane pores, the fraction of manganese particles slightly smaller than the pores in diameter was large. From these results, it is surmised that iron particles approximately the same size as the pores block them soon after the start of operation, and as the membrane pores narrow with the development of fouling, they become further blocked by manganese particles approximately the same size as the narrowed pores. Calcium and silica are assumed to accumulate on the membrane due to their cross-linking action and/or complex formation with organic substances such as humic compounds. The present research is the first to clearly show that the inorganic components that contribute to membrane fouling differ according to the stage of membrane fouling progression; the information obtained by this research should enable chemical cleaning or operational control in accordance with the stage of membrane fouling progression. Copyright © 2015 Elsevier Ltd. All rights reserved.

Design of a Tool Integrating Force Sensing With Automated Insertion in Cochlear Implantation

PubMed Central

Schurzig, Daniel; Labadie, Robert F.; Hussong, Andreas; Rau, Thomas S.; Webster, Robert J.

2012-01-01

The quality of hearing restored to a deaf patient by a cochlear implant in hearing preservation cochlear implant surgery (and possibly also in routine cochlear implant surgery) is believed to depend on preserving delicate cochlear membranes while accurately inserting an electrode array deep into the spiral cochlea. Membrane rupture forces, and possibly, other indicators of suboptimal placement, are below the threshold detectable by human hands, motivating a force sensing insertion tool. Furthermore, recent studies have shown significant variability in manual insertion forces and velocities that may explain some instances of imperfect placement. Toward addressing this, an automated insertion tool was recently developed by Hussong et al. By following the same insertion tool concept, in this paper, we present mechanical enhancements that improve the surgeon’s interface with the device and make it smaller and lighter. We also present electomechanical design of new components enabling integrated force sensing. The tool is designed to be sufficiently compact and light that it can be mounted to a microstereotactic frame for accurate image-guided preinsertion positioning. The new integrated force sensing system is capable of resolving forces as small as 0.005 N, and we provide experimental illustration of using forces to detect errors in electrode insertion. PMID:23482414

Multiplex lithography for multilevel multiscale architectures and its application to polymer electrolyte membrane fuel cell

NASA Astrophysics Data System (ADS)

Cho, Hyesung; Moon Kim, Sang; Sik Kang, Yun; Kim, Junsoo; Jang, Segeun; Kim, Minhyoung; Park, Hyunchul; Won Bang, Jung; Seo, Soonmin; Suh, Kahp-Yang; Sung, Yung-Eun; Choi, Mansoo

2015-09-01

The production of multiscale architectures is of significant interest in materials science, and the integration of those structures could provide a breakthrough for various applications. Here we report a simple yet versatile strategy that allows for the LEGO-like integrations of microscale membranes by quantitatively controlling the oxygen inhibition effects of ultraviolet-curable materials, leading to multilevel multiscale architectures. The spatial control of oxygen concentration induces different curing contrasts in a resin allowing the selective imprinting and bonding at different sides of a membrane, which enables LEGO-like integration together with the multiscale pattern formation. Utilizing the method, the multilevel multiscale Nafion membranes are prepared and applied to polymer electrolyte membrane fuel cell. Our multiscale membrane fuel cell demonstrates significant enhancement of performance while ensuring mechanical robustness. The performance enhancement is caused by the combined effect of the decrease of membrane resistance and the increase of the electrochemical active surface area.

Step-by-step seeding procedure for preparing HKUST-1 membrane on porous α-alumina support.

PubMed

Nan, Jiangpu; Dong, Xueliang; Wang, Wenjin; Jin, Wanqin; Xu, Nanping

2011-04-19

Metal-organic framework (MOF) membranes have attracted considerable attention because of their striking advantages in small-molecule separation. The preparation of an integrated MOF membrane is still a major challenge. Depositing a uniform seed layer on a support for secondary growth is a main route to obtaining an integrated MOF membrane. A novel seeding method to prepare HKUST-1 (known as Cu(3)(btc)(2)) membranes on porous α-alumina supports is reported. The in situ production of the seed layer was realized in step-by-step fashion via the coordination of H(3)btc and Cu(2+) on an α-alumina support. The formation process of the seed layer was observed by ultraviolet-visible absorption spectroscopy and atomic force microscopy. An integrated HKUST-1 membrane could be synthesized by the secondary hydrothermal growth on the seeded support. The gas permeation performance of the membrane was evaluated. © 2011 American Chemical Society

Regulation of multispanning membrane protein topology via post-translational annealing.

PubMed

Van Lehn, Reid C; Zhang, Bin; Miller, Thomas F

2015-09-26

The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

Dietary fatty acids and membrane protein function.

PubMed

Murphy, M G

1990-02-01

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

PubMed Central

Xiao, Z; Devreotes, P N

1997-01-01

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures. Images PMID:9168471

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

PubMed

Xiao, Z; Devreotes, P N

1997-05-01

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.

Resistance to and killing by the sporicidal microbicide peracetic acid.

PubMed

Leggett, Mark J; Schwarz, J Spencer; Burke, Peter A; Mcdonnell, Gerald; Denyer, Stephen P; Maillard, Jean-Yves

2015-03-01

To elucidate the mechanisms of spore resistance to and killing by the oxidizing microbicide peracetic acid (PAA). Mutants of Bacillus subtilis lacking specific spore structures were used to identify resistance properties in spores and to understand the mechanism of action of PAA. We also assessed the effect of PAA treatment on a number of spore properties including heat tolerance, membrane integrity and germination. The spore coat is essential for spore PAA resistance as spores with defective coats were greatly sensitized to PAA treatment. Small acid-soluble spore proteins apparently provide no protection against PAA. Defects in spore germination, specifically in germination via the GerB and GerK but not the GerA germination receptors, as well as leakage of internal components suggest that PAA is active at the spore inner membrane. It is therefore likely that the inner membrane is the major site of PAA's sporicidal activity. PAA treatment targets the spore membrane, with some of its activity directed specifically against the GerB and GerK germination receptors. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Hydrodynamic mobility of a sphere moving on the centerline of an elastic tube

NASA Astrophysics Data System (ADS)

Daddi-Moussa-Ider, Abdallah; Lisicki, Maciej; Gekle, Stephan

2017-11-01

Elastic channels are an important component of many soft matter systems, in which hydrodynamic interactions with confining membranes determine the behavior of particles in flow. In this work, we derive analytical expressions for Green's functions associated with a point-force (Stokeslet) directed parallel or perpendicular to the axis of an elastic cylindrical channel exhibiting resistance against shear and bending. We then compute the leading order self- and pair mobility functions of particles on the cylinder axis, finding that the mobilities are primarily determined by membrane shear and that bending does not play a significant role. In the quasi-steady limit of vanishing frequency, the particle self- and pair mobilities near a no-slip hard cylinder are recovered only if the membrane possesses a non-vanishing shear rigidity. We further compute the membrane deformation, finding that deformation is generally more pronounced in the axial (radial) directions, for the motion along (perpendicular to) the cylinder centerline, respectively. Our analytical calculations for Green's functions in an elastic cylinder can serve as a fundamental building block for future studies and are verified by fully resolved boundary integral simulations where very good agreement is obtained.

Electron tomography of early melanosomes: Implications for melanogenesis and the generation of fibrillar amyloid sheets

PubMed Central

Hurbain, Ilse; Geerts, Willie J. C.; Boudier, Thomas; Marco, Sergio; Verkleij, Arie J.; Marks, Michael S.; Raposo, Graç

2008-01-01

Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models. PMID:19033461

Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets.

PubMed

Hurbain, Ilse; Geerts, Willie J C; Boudier, Thomas; Marco, Sergio; Verkleij, Arie J; Marks, Michael S; Raposo, Graç

2008-12-16

Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.

Anti-Candida activity of geraniol involves disruption of cell membrane integrity and function.

PubMed

Sharma, Y; Khan, L A; Manzoor, N

2016-09-01

Candidiasis is a major problem in immunocompromised patients. Candida, an opportunistic fungal pathogen, is a major health concern today as conventional drugs are highly toxic with undesirable side effects. Their fungistatic nature is responsible for drug resistance in continuously evolving strains. Geraniol, an acyclic monoterpene alcohol, is a component of several plant essential oils. In the present study, an attempt has been made to understand the antifungal activity of geraniol at the cell membrane level in three Candida species. With an MIC of 30-130μg/mL, this natural compound was fungicidal at concentrations 2×MIC. There was complete suppression of fungal growth at MIC values (growth curves) and encouragingly geraniol is non-toxic even at the concentrations approaching 5×MIC (hemolysis assay). Exposed cells showed altered morphology, wherein the cells appeared either broken or shrivelled up (SEM studies). Significant reduction was seen in ergosterol levels at sub-MIC and glucose-induced H(+) efflux at concentrations>MIC values. Our results suggest that geraniol disrupts cell membrane integrity by interfering with ergosterol biosynthesis and inhibiting the very crucial PM-ATPase. It may hence be used in the management and treatment of both superficial and invasive candidiasis but further studies are required to elaborate its mode of action. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Screening anti-allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method.

PubMed

Lv, Yanni; Sun, Yueming; Fu, Jia; Kong, Liyun; Han, Shengli

2017-02-01

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti-allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method was developed to screen, analyze and identify the anti-allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC-ESI-MS/MS. The coupled system was then used to screen anti-allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose-dependent manner from 1 to 100 μm. The LAD2 cell membrane chromatography online with UHPLC-ESI-MS/MS method is an effective technique for screening the anti-allergic components of Huangqi. Copyright © 2016 John Wiley & Sons, Ltd.

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA.

PubMed

Mitrea, Diana M; Cika, Jaclyn A; Guy, Clifford S; Ban, David; Banerjee, Priya R; Stanley, Christopher B; Nourse, Amanda; Deniz, Ashok A; Kriwacki, Richard W

2016-02-02

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.

Design of a Novel Two-Component Hybrid Dermal Scaffold for the Treatment of Pressure Sores.

PubMed

Sharma, Vaibhav; Kohli, Nupur; Moulding, Dale; Afolabi, Halimat; Hook, Lilian; Mason, Chris; García-Gareta, Elena

2017-11-01

The aim of this study is to design a novel two-component hybrid scaffold using the fibrin/alginate porous hydrogel Smart Matrix combined to a backing layer of plasma polymerized polydimethylsiloxane (Sil) membrane to make the fibrin-based dermal scaffold more robust for the treatment of the clinically challenging pressure sores. A design criteria are established, according to which the Sil membranes are punched to avoid collection of fluid underneath. Manual peel test shows that native silicone does not attach to the fibrin/alginate component while the plasma polymerized silicone membranes are firmly bound to fibrin/alginate. Structural characterization shows that the fibrin/alginate matrix is intact after the addition of the Sil membrane. By adding a Sil membrane to the original fibrin/alginate scaffold, the resulting two-component scaffolds have a significantly higher shear or storage modulus G'. In vitro cell studies show that dermal fibroblasts remain viable, proliferate, and infiltrate the two-component hybrid scaffolds during the culture period. These results show that the design of a novel two-component hybrid dermal scaffold is successful according to the proposed design criteria. To the best of the authors' knowledge, this is the first study that reports the combination of a fibrin-based scaffold with a plasma-polymerized silicone membrane. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis and Characterisation of ETS-10/Acetate-based Ionic Liquid/Chitosan Mixed Matrix Membranes for CO2/N2 Permeation

PubMed Central

Casado-Coterillo, Clara; López-Guerrero, María del Mar; Irabien, Ángel

2014-01-01

Mixed matrix membranes (MMMs) were prepared by incorporating organic surfactant-free hydrothermally synthesised ETS-10 and 1-ethyl-3-methylimidazolium acetate ionic liquid (IL) to chitosan (CS) polymer matrix. The membrane material characteristics and permselectivity performance of the two-component membranes were compared with the three-component membrane and the pure CS membrane. The addition of IL increased CO2 solubility of the polymer, and, thus, the CO2 affinity was maintained for the MMMs, which can be correlated with the crystallinity, measured by FT-IR, and void fraction calculations from differences between theoretical and experimental densities. The mechanical resistance was enhanced by the ETS-10 nanoparticles, and flexibility decreased in the two-component ETS-10/CS MMMs, but the flexibility imparted by the IL remained in three-component ETS-10/IL/CS MMMs. The results of this work provide insight into another way of facing the adhesion challenge in MMMs and obtain CO2 selective MMMs from renewable or green chemistry materials. PMID:24957178

The correlation between biofilm biopolymer composition and membrane fouling in submerged membrane bioreactors.

PubMed

Luo, Jinxue; Zhang, Jinsong; Tan, Xiaohui; McDougald, Diane; Zhuang, Guoqiang; Fane, Anthony G; Kjelleberg, Staffan; Cohen, Yehuda; Rice, Scott A

2014-10-01

Biofouling, the combined effect of microorganism and biopolymer accumulation, significantly reduces the process efficiency of membrane bioreactors (MBRs). Here, four biofilm components, alpha-polysaccharides, beta-polysaccharides, proteins and microorganisms, were quantified in MBRs. The biomass of each component was positively correlated with the transmembrane pressure increase in MBRs. Proteins were the most abundant biopolymer in biofilms and showed the fastest rate of increase. The spatial distribution and co-localization analysis of the biofouling components indicated at least 60% of the extracellular polysaccharide (EPS) components were associated with the microbial cells when the transmembrane pressure (TMP) entered the jump phase, suggesting that the EPS components were either secreted by the biofilm cells or that the deposition of these components facilitated biofilm formation. It is suggested that biofilm formation and the accumulation of EPS are intrinsically coupled, resulting in biofouling and loss of system performance. Therefore, strategies that control biofilm formation on membranes may result in a significant improvement of MBR performance.

Specific Adhesion of Lipid Membranes Can Simultaneously Produce Two Types of Lipid and Protein Heterogeneities

NASA Astrophysics Data System (ADS)

Shindell, Orrin; Micah, Natalie; Ritzer, Max; Gordon, Vernita

2015-03-01

Living cells adhere to one another and their environment. Adhesion is associated with re-organization of the lipid and protein components of the cell membrane. The resulting heterogeneities are functional structures involved in biological processes. We use artificial lipid membranes that contain a single type of binding protein. Before adhesion, the lipid, protein, and dye components in the membrane are well-mixed and constitute a single disordered-liquid phase (Ld) . After adhesion, two distinct types of heterogeneities coexist in the adhesion zone: a central domain of ordered lipid phase that excludes both binding proteins and membrane dye, and a peripheral domain of disordered lipid phase that is densely packed with adhesion proteins and enriched in membrane dye relative to the non-adhered portion of the vesicle. Thus, we show that adhesion that is mediated by only one type of protein can organize the lipid and protein components of the membranes into heterogeneities that resemble those found in biology, for example the immune synapse.

Impact of oxLDL on Cholesterol-Rich Membrane Rafts

PubMed Central

Levitan, Irena; Shentu, Tzu-Pin

2011-01-01

Numerous studies have demonstrated that cholesterol-rich membrane rafts play critical roles in multiple cellular functions. However, the impact of the lipoproteins on the structure, integrity and cholesterol composition of these domains is not well understood. This paper focuses on oxidized low-density lipoproteins (oxLDLs) that are strongly implicated in the development of the cardiovascular disease and whose impact on membrane cholesterol and on membrane rafts has been highly controversial. More specifically, we discuss three major criteria for the impact of oxLDL on membrane rafts: distribution of different membrane raft markers, changes in membrane cholesterol composition, and changes in lipid packing of different membrane domains. We also propose a model to reconcile the controversy regarding the relationship between oxLDL, membrane cholesterol, and the integrity of cholesterol-rich membrane domains. PMID:21490811

Advanced, Energy-Efficient Hybrid Membrane System for Industrial Water Reuse

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

Toy, Lora; Choi, Young Chul; Hendren, Zachary

In the U.S. manufacturing sector, current industrial water use practices are energy-intensive and utilize and discharge high volumes of waters, rendering them not sustainable especially in light of the growing scarcity of suitable water supplies. To help address this problem, the goal of this project was to develop an advanced, cost-effective, hybrid membrane-based water treatment system that can improve the energy efficiency of industrial wastewater treatment while allowing at least 50% water reuse efficiency. This hybrid process would combine emerging Forward Osmosis (FO) and Membrane Distillation (MD) technology components into an integrated FO-MD system that can beneficially utilize low-grade wastemore » heat (i.e., T < 450 °F) in industrial facilities to produce distilled-quality product water for reuse. In this project, laboratory-, bench-, and pilot-scale experiments on the hybrid FO-MD system were conducted for industrial wastewater treatment. It was demonstrated at laboratory, bench, and pilot scales that FO-MD membrane technology can concentrate brine to very high total dissolved solids (TDS) levels (>200,000 ppm) that are at least 2.5 times higher than the TDS level to which RO can achieve. In laboratory testing, currently available FO and MD membranes were tested to select for high-performing membranes with high salt rejection and high water flux. Multiple FO membrane/draw-salt solution combinations that gave high water flux with higher than 98% salt rejection were also identified. Reverse draw-salt fluxes were observed to be much lower for divalent salts than for monovalent salts. MD membranes were identified that had 99.9+% salt rejection and water flux as high as 50-90 L/(m 2·h) for flat-sheet membranes and >20 L/(m 2·h) for hollow fibers. In bench-scale testing, a single unit of commercially available FO and MD membrane modules were evaluated for continuous, integrated operation. Using the laboratory- and bench-scale test data, numerical modeling was performed on the FO and MD processes to estimate engineering parameters for a larger-scale pilot unit. Based on the experimental studies and modeling results, a pilot-scale, integrated FO-MD prototype unit was designed and built for trailer-mounted operation. This prototype system was fed real industrial wastewater, which could not be further treated by conventional technologies, from an oil production facility and was successfully operated for over 15 weeks without major stoppage. About 90% water recovery was possible, while concentrating the TDS from 12,000 ppm up to 190,500 ppm. The FO-MD prototype rejected most wastewater contaminants while producing water with <300 ppm TDS, even when the feed TDS was higher than 150,000 ppm. No chemical cleaning was necessary during the pilot testing period. Flushing the system with dechlorinated tap water was sufficient to reset the membranes for the next set of test conditions. Pilot performance and membrane autopsy showed that, even though the feed was concentrated more than 10 times, membrane fouling was unnoticeable and no defects were detected on the FO and MD membrane surfaces. This project demonstrated the technical feasibility of the hybrid FO-MD process by taking water already treated to the limit with the highest level of current technologies and further concentrating it 10-fold by using mostly low-cost materials. Because no membranes suitable for full-scale plant applications are available at present, economical feasibility of the hybrid technology is still uncertain, but it is expected that broader industry participation can further reduce FO-MD process costs.« less

Reduced cholesterol levels in renal membranes of undernourished rats may account for urinary Na⁺ loss.

PubMed

Oliveira, Fabiana S T; Vieira-Filho, Leucio D; Cabral, Edjair V; Sampaio, Luzia S; Silva, Paulo A; Carvalho, Vera C O; Vieyra, Adalberto; Einicker-Lamas, Marcelo; Lima, Vera L M; Paixão, Ana D O

2013-04-01

It has been demonstrated that reabsorption of Na⁺ in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na⁺ loss and the inability to concentrate urine, were investigated in undernourished rats. Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na⁺ transporters, (Na⁺ + K⁺)ATPase and Na⁺-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration. Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na⁺ + K⁺)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na⁺-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na⁺ excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls. Life-long undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na⁺ + K⁺)ATPase activity resulting in reduced Na⁺ reabsorption and urinary concentrating ability.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

PubMed

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong; Gould, Kathleen L

2017-09-15

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

PubMed Central

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong

2017-01-01

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0–80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80–160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160–350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function. PMID:28914606

High throughput platforms for structural genomics of integral membrane proteins.

PubMed

Mancia, Filippo; Love, James

2011-08-01

Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

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

Kita, Ayako; Higa, Mari; Doi, Akira

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis withmore » multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.« less

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Biochemical and immunochemical analyses of detergent solubilized antigens from membrane vesicles of Aspergillus fumigatus.

PubMed

Piechura, J E; Riefel, R S; Daft, L J

1987-11-01

A membrane vesicle fraction isolated from exponentially growing Aspergillus fumigatus strain Ag 507 cultures was obtained by mechanical disruption of intact Aspergillus cells under specific osmotic conditions followed by a pH fractionation technique. Electron micrographs of the membrane vesicles indicated unit membrane structures free from cell wall material. High glucose-6-phosphatase and low lactate dehydrogenase activities verified the relative purity of the membrane vesicle fraction. Allergic bronchopulmonary aspergillosis (ABPA) patient and normal human sera were incubated with the membrane vesicle fraction followed by colloidal gold tagged rabbit antiserum to human IgG or IgE. Electron micrographs indicated ABPA patient sera possessed specific IgG and IgE antibodies to membranous components. The detergent octyl-beta-D-glucopyranoside was used to extract membrane vesicle components (MC). The enzyme profile of MC compared with cell sap components (CS) showed differences in types of enzymes. Two-dimensional polyacrylamide gel electrophoretic analyses of MC and CS detected components shared as well as unique to each fraction. In crossed immunoelectrophoresis using both rabbit antisera raised to MC and ABPA patient sera, 5 peaks were detected, while analysis of CS using rabbit antisera raised to CS produced 20 major peaks. Immunoelectrophoresis and double immunodiffusion data supported the crossed immunoelectrophoretic data: MC differed from CS. Enzyme-linked immunosorbent assay indicated high specific IgG and IgE antibody levels to MC in ABPA patient sera. Crossed immuno-affinoelectrophoresis with concanavalin A partially characterized the MC, which consist of components which have glycoprotein elements (i.e., containing alpha-D-glucose or alpha-D-mannose).

Boundary-integral modeling of cochlear hydrodynamics

NASA Astrophysics Data System (ADS)

Pozrikidis, C.

2008-04-01

A two-dimensional model that captures the essential features of the vibration of the basilar membrane of the cochlea is proposed. The flow due to the vibration of the stapes footplate and round window is modeled by a point source and a point sink, and the cochlear pressure is computed simultaneously with the oscillations of the basilar membrane. The mathematical formulation relies on the boundary-integral representation of the potential flow established far from the basilar membrane and cochlea side walls, neglecting the thin Stokes boundary layer lining these surfaces. The boundary-integral approach furnishes integral equations for the membrane vibration amplitude and pressure distribution on the upper or lower side of the membrane. Several approaches are discussed, and numerical solutions in the frequency domain are presented for a rectangular cochlea model using different membrane response functions. The numerical results reproduce and extend the theoretical predictions of previous authors and delineate the effect of physical and geometrical parameters. It is found that the membrane vibration depends weakly on the position of the membrane between the upper and lower wall of the cochlear channel and on the precise location of the oval and round windows. Solutions of the initial-value problem with a single-period sinusoidal impulse reveal the formation of a traveling wave packet that eventually disappears at the helicotrema.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

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

Intermolecular interactions at early stage of protein/detergent particle association induced by salt/polyethylene glycol mixtures.

PubMed

Odahara, Takayuki; Odahara, Koji

2016-04-01

Mixtures of neutral salts and polyethylene glycol are used for various purposes in biological studies. Although the effects of each component of the mixtures are theoretically well investigated, comprehension of their integrated effects remains insufficient. In this work, their roles and effects as a precipitant were clarified by studying dependence of precipitation curves on salt concentration for integral membrane protein/detergent particles of different physicochemical properties. The dependence of precipitation curves was reasonably related to intermolecular interactions among relevant molecules such as protein, detergent and polyethylene glycol by considering their physicochemical properties. The obtained relationships are useful as basic information to learn the early stage of biological macromolecular associations. Copyright © 2015 Elsevier Inc. All rights reserved.

Recycling of used perfluorosulfonic acid membranes

DOEpatents

Grot, Stephen [Middletown, DE; Grot, Walther [Chadds Ford, PA

2007-08-14

A method for recovering and recycling catalyst coated fuel cell membranes includes dissolving the used membranes in water and solvent, heating the dissolved membranes under pressure and separating the components. Active membranes are produced from the recycled materials.

Integrating seawater desalination and wastewater reclamation forward osmosis process using thin-film composite mixed matrix membrane with functionalized carbon nanotube blended polyethersulfone support layer.

PubMed

Choi, Hyeon-Gyu; Son, Moon; Choi, Heechul

2017-10-01

Thin-film composite mixed matrix membrane (TFC MMM) with functionalized carbon nanotube (fCNT) blended in polyethersulfone (PES) support layer was synthesized via interfacial polymerization and phase inversion. This membrane was firstly tested in lab-scale integrating seawater desalination and wastewater reclamation forward osmosis (FO) process. Water flux of TFC MMM was increased by 72% compared to that of TFC membrane due to enhanced hydrophilicity. Although TFC MMM showed lower water flux than TFC commercial membrane, enhanced reverse salt flux selectivity (RSFS) of TFC MMM was observed compared to TFC membrane (15% higher) and TFC commercial membrane (4% higher), representing membrane permselectivity. Under effluent organic matter (EfOM) fouling test, 16% less normalized flux decline of TFC MMM was observed compared to TFC membrane. There was 8% less decline of TFC MMM compared to TFC commercial membrane due to fCNT effect on repulsive foulant-membrane interaction enhancement, caused by negatively charged membrane surface. After 10 min physical cleaning, TFC MMM displayed higher recovered normalized flux than TFC membrane (6%) and TFC commercial membrane (4%); this was also supported by visualized characterization of fouling layer. This study presents application of TFC MMM to integrated seawater desalination and wastewater reclamation FO process for the first time. It can be concluded that EfOM fouling of TFC MMM was suppressed due to repulsive foulant-membrane interaction. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multiplex lithography for multilevel multiscale architectures and its application to polymer electrolyte membrane fuel cell

PubMed Central

Cho, Hyesung; Moon Kim, Sang; Sik Kang, Yun; Kim, Junsoo; Jang, Segeun; Kim, Minhyoung; Park, Hyunchul; Won Bang, Jung; Seo, Soonmin; Suh, Kahp-Yang; Sung, Yung-Eun; Choi, Mansoo

2015-01-01

The production of multiscale architectures is of significant interest in materials science, and the integration of those structures could provide a breakthrough for various applications. Here we report a simple yet versatile strategy that allows for the LEGO-like integrations of microscale membranes by quantitatively controlling the oxygen inhibition effects of ultraviolet-curable materials, leading to multilevel multiscale architectures. The spatial control of oxygen concentration induces different curing contrasts in a resin allowing the selective imprinting and bonding at different sides of a membrane, which enables LEGO-like integration together with the multiscale pattern formation. Utilizing the method, the multilevel multiscale Nafion membranes are prepared and applied to polymer electrolyte membrane fuel cell. Our multiscale membrane fuel cell demonstrates significant enhancement of performance while ensuring mechanical robustness. The performance enhancement is caused by the combined effect of the decrease of membrane resistance and the increase of the electrochemical active surface area. PMID:26412619

Development of new dyes for use in integrated optical sensors.

PubMed

Citterio, D; Rásonyi, S; Spichiger, U E

1996-03-01

New chromoionophores have been developed, focused on NIR applications so that optode membranes may be used in monolithically integrated optical sensors. The wavelength of maximum absorbance has been estimated for a new model compound by the Pariser-Parr-Pople (PPP) method. Several cyanine type dyes have been tested as membrane chromoionophores. Membrane composition has been altered to overcome solubility problems. In this way, simple pH-sensitive optode membranes have been produced.

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability**

PubMed Central

Swainsbury, David J K; Scheidelaar, Stefan; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2014-01-01

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. PMID:25212490

The fine art of integral membrane protein crystallisation.

PubMed

Birch, James; Axford, Danny; Foadi, James; Meyer, Arne; Eckhardt, Annette; Thielmann, Yvonne; Moraes, Isabel

2018-05-18

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation. Copyright © 2018 Diamond Light Source LTD. Published by Elsevier Inc. All rights reserved.

Computational Insight Into the Structural Organization of Full-Length Toll-Like Receptor 4 Dimer in a Model Phospholipid Bilayer

PubMed Central

Patra, Mahesh Chandra; Kwon, Hyuk-Kwon; Batool, Maria; Choi, Sangdun

2018-01-01

Toll-like receptors (TLRs) are a unique category of pattern recognition receptors that recognize distinct pathogenic components, often utilizing the same set of downstream adaptors. Specific molecular features of extracellular, transmembrane (TM), and cytoplasmic domains of TLRs are crucial for coordinating the complex, innate immune signaling pathway. Here, we constructed a full-length structural model of TLR4—a widely studied member of the interleukin-1 receptor/TLR superfamily—using homology modeling, protein–protein docking, and molecular dynamics simulations to understand the differential domain organization of TLR4 in a membrane-aqueous environment. Results showed that each functional domain of the membrane-bound TLR4 displayed several structural transitions that are biophysically essential for plasma membrane integration. Specifically, the extracellular and cytoplasmic domains were partially immersed in the upper and lower leaflets of the membrane bilayer. Meanwhile, TM domains tilted considerably to overcome the hydrophobic mismatch with the bilayer core. Our analysis indicates an alternate dimerization or a potential oligomerization interface of TLR4-TM. Moreover, the helical properties of an isolated TM dimer partly agree with that of the full-length receptor. Furthermore, membrane-absorbed or solvent-exposed surfaces of the toll/interleukin-1 receptor domain are consistent with previous X-ray crystallography and biochemical studies. Collectively, we provided a complete structural model of membrane-bound TLR4 that strengthens our current understanding of the complex mechanism of receptor activation and adaptor recruitment in the innate immune signaling pathway. PMID:29593733

Composite membrane with integral rim

DOEpatents

Routkevitch, Dmitri; Polyakov, Oleg G

2015-01-27

Composite membranes that are adapted for separation, purification, filtration, analysis, reaction and sensing. The composite membranes can include a porous support structure having elongate pore channels extending through the support structure. The composite membrane also includes an active layer comprising an active layer material, where the active layer material is completely disposed within the pore channels between the surfaces of the support structure. The active layer is intimately integrated within the support structure, thus enabling great robustness, reliability, resistance to mechanical stress and thermal cycling, and high selectivity. Methods for the fabrication of composite membranes are also provided.

Mechanism Design and Testing of a Self-Deploying Structure Using Flexible Composite Tape Springs

NASA Technical Reports Server (NTRS)

Footdale, Joseph N.; Murphey, Thomas W.

2014-01-01

The detailed mechanical design of a novel deployable support structure that positions and tensions a membrane optic for space imagining applications is presented. This is a complex three-dimensional deployment using freely deploying rollable composite tape spring booms that become load bearing structural members at full deployment. The deployment tests successfully demonstrate a new architecture based on rolled and freely deployed composite tape spring members that achieve simultaneous deployment without mechanical synchronization. Proper design of the flexible component mounting interface and constraint systems, which were critical in achieving a functioning unit, are described. These flexible composite components have much potential for advancing the state of the art in deployable structures, but have yet to be widely adopted. This paper demonstrates the feasibility and advantages of implementing flexible composite components, including the design details on how to integrate with required traditional mechanisms.

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

PubMed Central

Orlean, Peter

2012-01-01

The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

Depot effect of bioactive components in experimental membrane filtrations

NASA Astrophysics Data System (ADS)

Mitev, D.; Peshev, D.; Peev, G.; Peeva, L.

2017-01-01

Depot effects were found to be accompanying phenomena of membrane separation processes. Accumulation of target species in the membrane matrix during feasibility tests can hamper proper conclusions or compromise the filtration results. Therefore, we investigated the effects of delayed membrane release of chlorogenic acid and caffeine, considered as key compounds of interest in spent coffee products’ recovery treatment. Permeate fluxes and key components release were studied in course of 24 hours via nanofiltration of pure solvent, both immediately after the mock solution filtration and after idle stay. Conclusions are drawn and recommendations advised for proper analysis of experimental data on membrane screening.

Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays.

PubMed

Ghosh, Madhubrata; Wang, Loo Chien; Ramesh, Ranita; Morgan, Leslie K; Kenney, Linda J; Anand, Ganesh S

2017-02-28

Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Pervaporation behavior and integrated process for concentrating lignocellulosic ethanol through polydimethylsiloxane (PDMS) membrane.

PubMed

Chen, Jingwen; Zhang, Hongman; Wei, Ping; Zhang, Lin; Huang, He

2014-02-01

The effects of by-products from ethanol fermentation and hydrolysates of lignocelluloses on ethanol diffusion through polydimethylsiloxane (PDMS) membranes with/without silicalite-1 were investigated. A pervaporation process was integrated with lignocellulosic fermentation to concentrate bioethanol using bare PDMS membranes. Results showed that yeasts, solid particles, and salts increased ethanol flux and selectivity through the membranes (PDMS with/without silicalite-1), whereas glucose exerted negative effects on the performance. On bare PDMS membrane, the performance was not obviously affected by the existence of aliphatic acids. However, on PDMS-silicalite-1 membrane, a remarkable decrease in ethanol selectivity and a rapid growth of total flux in the presence of aliphatic acids were observed. These phenomena were due to the interaction of acids with silanol (Si-OH) groups to break the dense membrane surface. On the PDMS membranes with/without silicalite-1, degradation products of lignocellulosic hydrolysates such as furfural and hydroxyacetone slightly influenced separation performance. These results revealed that an integrated process can effectively eliminate product inhibition, improve ethanol productivity, and enhance the glucose conversion rate.

Gradient composite metal-ceramic foam as supportive component for planar SOFCs and MIEC membranes

NASA Astrophysics Data System (ADS)

Smorygo, Oleg; Mikutski, Vitali; Marukovich, Alexander; Sadykov, Vladislav; Usoltsev, Vladimir; Mezentseva, Natalia; Borodinecs, Anatolijs; Bobrenok, Oleg

2011-06-01

A novel approach to the design of planar gradient porous supports for the thin-film SOFCs and MIEC membranes is described. The support's thermal expansion is controlled by the creation of a two-component composite metal-ceramic foam structure. Thin MIEC membranes and SOFCs were prepared on the composite supports by the layerwise deposition of composite functional layers including complex fluorites and perovskites. Lab-scale studies demonstrated promising performance of both MIEC membrane and SOFC.

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

Regulation of necrotic cell death p53, PARP1 and Cyclophilin D -overlapping pathways of regulated necrosis?

PubMed Central

Ying, Yuan; Padanilam, Babu J.

2017-01-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator (ANT) and the phosphate carrier (PiC) are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury. PMID:27048819

Regulation of necrotic cell death: p53, PARP1 and cyclophilin D-overlapping pathways of regulated necrosis?

PubMed

Ying, Yuan; Padanilam, Babu J

2016-06-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator and the phosphate carrier are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury.

A novel requirement for C. elegans Alix/ALX-1 in RME-1 mediated membrane transport

PubMed Central

Shi, Anbing; Pant, Saumya; Balklava, Zita; Chen, Carlos Chih-Hsiung; Figueroa, Vanesa; Grant, Barth D.

2007-01-01

Summary Background Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition Alix is associated with the actin cytoskeleton and may regulate cytoskeletal dynamics. Results Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane called RME-1. Analysis of alx-1 mutants indicates that ALX-1 is required for endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by analysis of rme-1 mutants. Expression of truncated human Alix in HeLa cells disrupts recycling of MHCI, a known Ehd1/RME-1 dependent transport step, suggesting phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears dispensable for ALX-1 function in MVEs/late endosomes. Conclusions This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1. PMID:17997305

Dielectric behavior of beef meat in the 1-1500kHz range: Simulation with the Fricke/Cole-Cole model.

PubMed

Damez, Jean-Louis; Clerjon, Sylvie; Abouelkaram, Saïd; Lepetit, Jacques

2007-12-01

The electrical properties of biological tissues have been researched for many years. Impedance measurements observed with increasing frequencies are mainly attributed to changes in membrane conductivity and ion and charged-molecule mobility (mainly Na(+), K(+), CL(-) ions). Equivalent circuits with passive electrical components are frequently used as a support model for presentation and analyses of the behavior of tissues submitted to electrical fields. Fricke proposed an electrical model where the elements are resistive and capacitive. The model is composed of a resistive element (Rp) representing extracellular fluids (ECF) placed in parallel with a capacitive element (Cs) representing insulating membranes in series and a resistive element (Rs) representing intracellular fluids (ICF). This model is able to describe impedance measurements: at lower frequencies, most of the current flows around the cells without being able to penetrate them, while at higher frequencies the membranes lose their insulating properties and the current flows through both the extracellular and intracellular compartments. Since meat ageing induces structural change, particularly in membrane integrity, the insulating properties of membranes decrease, and intracellular and extracellular electrolytes mix, thus driving changes in their electrical properties. We report a method combining the Fricke and Cole-Cole models that was developed to monitor and explain tissues conductivity changes in preferential directions during beef meat ageing.

Light-activated control of protein channel assembly mediated by membrane mechanics

NASA Astrophysics Data System (ADS)

Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

2016-12-01

Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis*♦

PubMed Central

Yao, Jiangwei; Cherian, Philip T.; Frank, Matthew W.; Rock, Charles O.

2015-01-01

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine. PMID:25995447

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

PubMed

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix

PubMed Central

Ayala-Figueroa, Jesus; Parry, Jean M.; Pu, Pu; Hall, David H.

2016-01-01

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity. PMID:27482894

Membrane materials for storing biological samples intended for comparative nanotoxicological testing

NASA Astrophysics Data System (ADS)

Metelkin, A.; Kuznetsov, D.; Kolesnikov, E.; Chuprunov, K.; Kondakov, S.; Osipov, A.; Samsonova, J.

2015-11-01

The study is aimed at identifying the samples of most promising membrane materials for storing dry specimens of biological fluids (Dried Blood Spots, DBS technology). Existing sampling systems using cellulose fiber filter paper have a number of drawbacks such as uneven distribution of the sample spot, dependence of the spot spreading area on the individual biosample properties, incomplete washing-off of the sample due to partially inconvertible sorption of blood components on cellulose fibers, etc. Samples of membrane materials based on cellulose, polymers and glass fiber with applied biosamples were studied using methods of scanning electron microscopy, FT-IR spectroscopy and surface-wetting measurement. It was discovered that cellulose-based membrane materials sorb components of biological fluids inside their structure, while membranes based on glass fiber display almost no interaction with the samples and biological fluid components dry to films in the membrane pores between the structural fibers. This characteristic, together with the fact that membrane materials based on glass fiber possess sufficient strength, high wetting properties and good storage capacity, attests them as promising material for dry samples of biological fluids storage systems.

Recovery of real dye bath wastewater using integrated membrane process: considering water recovery, membrane fouling and reuse potential of membranes.

PubMed

Balcik-Canbolat, Cigdem; Sengezer, Cisel; Sakar, Hacer; Karagunduz, Ahmet; Keskinler, Bulent

2017-11-01

It has been recognized by the whole world that textile industry which produce large amounts of wastewater with strong color and toxic organic compounds is a major problematical industry requiring effective treatment solutions. In this study, reverse osmosis (RO) membranes were tested on biologically treated real dye bath wastewater with and without pretreatment by nanofiltration (NF) membrane to recovery. Also membrane fouling and reuse potential of membranes were investigated by multiple filtrations. Obtained results showed that only NF is not suitable to produce enough quality to reuse the wastewater in a textile industry as process water while RO provide successfully enough permeate quality. The results recommend that integrated NF/RO membrane process is able to reduce membrane fouling and allow long-term operation for real dye bath wastewater.

Novel polymer membrane process for pre-combustion CO{sub 2} capture from coal-fired syngas

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

Merkel, Tim

2011-09-14

This final report describes work conducted for the Department of Energy (DOE NETL) on development of a novel polymer membrane process for pre-combustion CO{sub 2} capture from coalfired syngas (award number DE-FE0001124). The work was conducted by Membrane Technology and Research, Inc. (MTR) from September 15, 2009, through December 14, 2011. Tetramer Technologies, LLC (Tetramer) was our subcontract partner on this project. The National Carbon Capture Center (NCCC) at Wilsonville, AL, provided access to syngas gasifier test facilities. The main objective of this project was to develop a cost-effective membrane process that could be used in the relatively near-term tomore » capture CO{sub 2} from shifted syngas generated by a coal-fired Integrated Gasification Combined Cycle (IGCC) power plant. In this project, novel polymeric membranes (designated as Proteus™ membranes) with separation properties superior to conventional polymeric membranes were developed. Hydrogen permeance of up to 800 gpu and H{sub 2}/CO{sub 2} selectivity of >12 was achieved using a simulated syngas mixture at 150°C and 50 psig, which exceeds the original project targets of 200 gpu for hydrogen permeance and 10 for H{sub 2}/CO{sub 2} selectivity. Lab-scale Proteus membrane modules (with a membrane area of 0.13 m{sup 2}) were also developed using scaled-up Proteus membranes and high temperature stable module components identified during this project. A mixed-gas hydrogen permeance of about 160 gpu and H{sub 2}/CO{sub 2} selectivity of >12 was achieved using a simulated syngas mixture at 150°C and 100 psig. We believe that a significant improvement in the membrane and module performance is likely with additional development work. Both Proteus membranes and lab-scale Proteus membrane modules were further evaluated using coal-derived syngas streams at the National Carbon Capture Center (NCCC). The results indicate that all module components, including the Proteus membrane, were stable under the field conditions (feed pressures: 150-175 psig and feed temperatures: 120-135°C) for over 600 hours. The field performance of both Proteus membrane stamps and Proteus membrane modules is consistent with the results obtained in the lab, suggesting that the presence of sulfur-containing compounds (up to 780 ppm hydrogen sulfide), saturated water vapor, carbon monoxide and heavy hydrocarbons in the syngas feed stream has no adverse effect on the Proteus membrane or module performance. We also performed an economic analysis for a number of membrane process designs developed in this project (using hydrogen-selective membranes, alone or in the combination with CO{sub 2}- selective membranes). The current field performance for Proteus membranes was used in the design analysis. The study showed the current best design has the potential to reduce the increase in Levelized Cost of Electricity (LCOE) caused by 90% CO{sub 2} capture to about 15% if co-sequestration of H{sub 2}S is viable. This value is still higher than the DOE target for increase in LCOE (10%); however, compared to the base-case Selexol process that gives a 30% increase in LCOE at 90% CO2 capture, the membrane-based process appears promising. We believe future improvements in membrane performance have the potential to reach the DOE target.« less

Intestinal epithelial wound healing assay in an epithelial-mesenchymal co-culture system.

PubMed

Seltana, Amira; Basora, Nuria; Beaulieu, Jean-François

2010-01-01

Rapid and efficient healing of epithelial damage is critical to the functional integrity of the small intestine. Epithelial repair is a complex process that has largely been studied in cultured epithelium but to a much lesser extent in mucosa. We describe a novel method for the study of wound healing using a co-culture system that combined an intestinal epithelial Caco-2/15 cell monolayer cultured on top of human intestinal myofibroblasts, which together formed a basement membrane-like structure that contained many of the major components found at the epithelial-mesenchymal interface in the human intestine. To investigate the mechanism of restitution, small lesions were generated in epithelial cell monolayers on plastic or in co-cultures without disturbing the underlying mesenchymal layer. Monitoring of wound healing showed that repair was more efficient in Caco-2/15-myofibroblast co-cultures than in Caco-2/15 monolayers and involved the deposition of basement membrane components. Functional experiments showed that the addition of type I collagen or human fibronectin to the culture medium significantly accelerated wound closure on epithelial cell co-cultures. This system may provide a new tool to investigate the mechanisms that regulate wound healing in the intestinal epithelium.

Integration of Ganglioside GT1b Receptor into DPPE and DPPC Phospholipid Monolayers: An X-Ray Reflectivity and Grazing-Incidence Diffraction Study

PubMed Central

Miller, C. E.; Busath, D. D.; Strongin, B.; Majewski, J.

2008-01-01

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT1b-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT1b is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT1b, the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23°C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT1b was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration. PMID:18599631

Nanoneedle insertion into the cell nucleus does not induce double-strand breaks in chromosomal DNA.

PubMed

Ryu, Seunghwan; Kawamura, Ryuzo; Naka, Ryohei; Silberberg, Yaron R; Nakamura, Noriyuki; Nakamura, Chikashi

2013-09-01

An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fluorescent Protein Aided Insights on Plastids and their Extensions: A Critical Appraisal

PubMed Central

Delfosse, Kathleen; Wozny, Michael R.; Jaipargas, Erica-Ashley; Barton, Kiah A.; Anderson, Cole; Mathur, Jaideep

2016-01-01

Multi-colored fluorescent proteins targeted to plastids have provided new insights on the dynamic behavior of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli, and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signaling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes. PMID:26834765

Integrated distillation-membrane process for bio-ethanol and bio-butanol recovery from actual fermentation broths: Separation energy efficiency and fate of secondary fermentation products

EPA Science Inventory

A hybrid process integrating vapor stripping with vapor compression and vapor permeation membrane separation, termed Membrane Assisted Vapor Stripping (MAVS), was evaluated for recovery and dehydration of ethanol and/or 1-butanol from aqueous solution as an alternative to convent...

Architecture, component, and microbiome of biofilm involved in the fouling of membrane bioreactors.

PubMed

Inaba, Tomohiro; Hori, Tomoyuki; Aizawa, Hidenobu; Ogata, Atsushi; Habe, Hiroshi

2017-01-01

Biofilm formation on the filtration membrane and the subsequent clogging of membrane pores (called biofouling) is one of the most persistent problems in membrane bioreactors for wastewater treatment and reclamation. Here, we investigated the structure and microbiome of fouling-related biofilms in the membrane bioreactor using non-destructive confocal reflection microscopy and high-throughput Illumina sequencing of 16S rRNA genes. Direct confocal reflection microscopy indicated that the thin biofilms were formed and maintained regardless of the increasing transmembrane pressure, which is a common indicator of membrane fouling, at low organic-loading rates. Their solid components were primarily extracellular polysaccharides and microbial cells. In contrast, high organic-loading rates resulted in a rapid increase in the transmembrane pressure and the development of the thick biofilms mainly composed of extracellular lipids. High-throughput sequencing revealed that the biofilm microbiomes, including major and minor microorganisms, substantially changed in response to the organic-loading rates and biofilm development. These results demonstrated for the first time that the architectures, chemical components, and microbiomes of the biofilms on fouled membranes were tightly associated with one another and differed considerably depending on the organic-loading conditions in the membrane bioreactor, emphasizing the significance of alternative indicators other than the transmembrane pressure for membrane biofouling.

Advanced imaging as a novel approach to the characterization of membranes for microfiltration applications

NASA Astrophysics Data System (ADS)

Marroquin, Milagro

The primary objectives of my dissertation were to design, develop and implement novel confocal microscopy imaging protocols for the characterization of membranes and highlight opportunities to obtain reliable and cutting-edge information of microfiltration membrane morphology and fouling processes. After a comprehensive introduction and review of confocal microscopy in membrane applications (Chapter 1), the first part of this dissertation (Chapter 2) details my work on membrane morphology characterization by confocal laser scanning microscopy (CLSM) and the implementation of my newly developed CLSM cross-sectional imaging protocol. Depth-of-penetration limits were identified to be approximately 24 microns and 7-8 microns for mixed cellulose ester and polyethersulfone membranes, respectively, making it impossible to image about 70% of the membrane bulk. The development and implementation of my cross-sectional CLSM method enabled the imaging of the entire membrane cross-section. Porosities of symmetric and asymmetric membranes with nominal pore sizes in the range 0.65-8.0 microns were quantified at different depths and yielded porosity values in the 50-60% range. It is my hope and expectation that the characterization strategy developed in this part of the work will enable future studies of different membrane materials and applications by confocal microscopy. After demonstrating how cross-sectional CLSM could be used to fully characterize membrane morphologies and porosities, I applied it to the characterization of fouling occurring in polyethersulfone microfiltration membranes during the processing of solutions containing proteins and polysaccharides (Chapter 3). Through CLSM imaging, it was determined where proteins and polysaccharides deposit throughout polymeric microfiltration membranes when a fluid containing these materials is filtered. CLSM enabled evaluation of the location and extent of fouling by individual components (protein: casein and polysaccharide: dextran) within wet, asymmetric polyethersulfone microfiltration membranes. Information from filtration flux profiles and cross-sectional CLSM images of the membranes that processed single-component solutions and mixtures agreed with each other. Concentration profiles versus depth for each individual component present in the feed solution were developed from the analysis of the CLSM images at different levels of fouling for single-component solutions and mixtures. CLSM provided visual information that helped elucidate the role of each component on membrane fouling and provided a better understanding of how component interactions impact the fouling profiles. Finally, Chapter 4 extends the application of my cross-sectional CLSM imaging protocol to study the fouling of asymmetric polyethersulfone membranes during the microfiltration of protein, polyphenol, and polysaccharide mixtures to better understand the solute-solute and solute-membrane interactions leading to fouling in beverage clarification processes. Again, cross-sectional CLSM imaging provided information on the location and extent of fouling throughout the entire thickness of the PES membrane. Quantitative analysis of the cross-sectional CLSM images provided a measurement of the masses of foulants deposited throughout the membrane. Moreover, flux decline data collected for different mixtures of casein, tannic acid and beta-cyclodextrin were analyzed with standard fouling models to determine the fouling mechanisms at play when processing different combinations of foulants. Results from model analysis of flux data were compared with the quantitative visual analysis of the correspondent CLSM images. This approach, which couples visual and performance measurements, is expected to provide a better understanding of the causes of fouling that, in turn, is expected to aid in the design of new membranes with tailored structure or surface chemistry that prevents the deposition of the foulants in "prone to foul" regions. (Abstract shortened by UMI.)

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

PubMed

Mossberg, Ann-Kristin; Puchades, Maja; Halskau, Øyvind; Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

2010-02-23

Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

Membrane-based systems for carbon capture and hydrogen purification

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

Berchtold, Kathryn A

2010-11-24

This presentation describes the activities being conducted at Los Alamos National Laboratory to develop carbon capture technologies for power systems. This work is aimed at continued development and demonstration of a membrane based pre- and post-combustion carbon capture technology and separation schemes. Our primary work entails the development and demonstration of an innovative membrane technology for pre-combustion capture of carbon dioxide that operates over a broad range of conditions relevant to the power industry while meeting the US DOE's Carbon Sequestration Program goals of 90% CO{sub 2} capture at less than a 10% increase in the cost of energy services.more » Separating and capturing carbon dioxide from mixed gas streams is a first and critical step in carbon sequestration. To be technically and economically viable, a successful separation method must be applicable to industrially relevant gas streams at realistic temperatures and pressures as well as be compatible with large gas volumes. Our project team is developing polymer membranes based on polybenzimidazole (PBI) chemistries that can purify hydrogen and capture CO{sub 2} at industrially relevant temperatures. Our primary objectives are to develop and demonstrate polymer-based membrane chemistries, structures, deployment platforms, and sealing technologies that achieve the critical combination of high selectivity, high permeability, chemical stability, and mechanical stability all at elevated temperatures (> 150 C) and packaged in a scalable, economically viable, high area density system amenable to incorporation into an advanced Integrated Gasification Combined-Cycle (IGCC) plant for pre-combustion CO{sub 2} capture. Stability requirements are focused on tolerance to the primary synthesis gas components and impurities at various locations in the IGCC process. Since the process stream compositions and conditions (temperature and pressure) vary throughout the IGCC process, the project is focused on the optimization of a technology that could be positioned upstream or downstream of one or more of the water-gas-shift reactors (WGSRs) or integrated with a WGSR.« less

Shared Components of Rhythm Generation for Locomotion and Scratching Exist Prior to Motoneurons

PubMed Central

Hao, Zhao-Zhe; Berkowitz, Ari

2017-01-01

Does the spinal cord use a single network to generate locomotor and scratching rhythms or two separate networks? Previous research showed that simultaneous swim and scratch stimulation (“dual stimulation”) in immobilized, spinal turtles evokes a single rhythm in hindlimb motor nerves with a frequency often greater than during swim stimulation alone or scratch stimulation alone. This suggests that the signals that trigger swimming and scratching converge and are integrated within the spinal cord. However, these results could not determine whether the integration occurs in motoneurons themselves or earlier, in spinal interneurons. Here, we recorded intracellularly from hindlimb motoneurons during dual stimulation. Motoneuron membrane potentials displayed regular oscillations at a higher frequency during dual stimulation than during swim or scratch stimulation alone. In contrast, arithmetic addition of the oscillations during swimming alone and scratching alone with various delays always generated irregular oscillations. Also, the standard deviation of the phase-normalized membrane potential during dual stimulation was similar to those during swimming or scratching alone. In contrast, the standard deviation was greater when pooling cycles of swimming alone and scratching alone for two of the three forms of scratching. This shows that dual stimulation generates a single rhythm prior to motoneurons. Thus, either swimming and scratching largely share a rhythm generator or the two rhythms are integrated into one rhythm by strong interactions among interneurons. PMID:28848402

Tripartite assembly of RND multidrug efflux pumps

NASA Astrophysics Data System (ADS)

Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

2016-02-01

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Tunable Microfluidic Devices for Hydrodynamic Fractionation of Cells and Beads: A Review

PubMed Central

Alvankarian, Jafar; Majlis, Burhanuddin Yeop

2015-01-01

The adjustable microfluidic devices that have been developed for hydrodynamic-based fractionation of beads and cells are important for fast performance tunability through interaction of mechanical properties of particles in fluid flow and mechanically flexible microstructures. In this review, the research works reported on fabrication and testing of the tunable elastomeric microfluidic devices for applications such as separation, filtration, isolation, and trapping of single or bulk of microbeads or cells are discussed. Such microfluidic systems for rapid performance alteration are classified in two groups of bulk deformation of microdevices using external mechanical forces, and local deformation of microstructures using flexible membrane by pneumatic pressure. The main advantage of membrane-based tunable systems has been addressed to be the high capability of integration with other microdevice components. The stretchable devices based on bulk deformation of microstructures have in common advantage of simplicity in design and fabrication process. PMID:26610519

Vibrational spectra of individual millimeter-size membrane patches using miniature infrared waveguides.

PubMed Central

Plunkett, S E; Jonas, R E; Braiman, M S

1997-01-01

We have used miniature planar IR waveguides, consisting of Ge strips 30-50 microm thick and 2 mm wide, as evanescent-wave sensors to detect the mid-(IR) evanescent-wave absorbance spectra of small areas of biomolecular monolayers and multilayers. Examples include picomolar quantities of an integral transmembrane protein (bacteriorhodopsin) and lipid (dimyristoyl phosphatidylcholine). IR bands due to the protein and lipid components of the plasma membrane of individual 1.5-mm-diameter devitellinized Xenopus laevis oocytes, submerged in buffer and sticking to the waveguide surface, were also detected. A significant improvement in sensitivity was observed, as compared to previous sizes and geometries of evanescent-wave sensors (e.g., commercially available internal reflection elements or tapered optical fibers). These measurements suggest the feasibility of using such miniature supported planar IR waveguides to observe structural changes in transmembrane proteins functioning in vivo in single cells. PMID:9336219

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

PubMed

Nordström, Viola; Willershäuser, Monja; Herzer, Silke; Rozman, Jan; von Bohlen Und Halbach, Oliver; Meldner, Sascha; Rothermel, Ulrike; Kaden, Sylvia; Roth, Fabian C; Waldeck, Clemens; Gretz, Norbert; de Angelis, Martin Hrabě; Draguhn, Andreas; Klingenspor, Martin; Gröne, Hermann-Josef; Jennemann, Richard

2013-01-01

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

Effluent Treatment Technologies in the Iron and Steel Industry - A State of the Art Review.

PubMed

Das, Pallabi; Mondal, Gautam C; Singh, Siddharth; Singh, Abhay K; Prasad, Bably; Singh, Krishna K

2018-05-01

  Iron and steel industry is the principal driving force propelling economic and technological growth of a nation. However, since its inception this industry is associated with widespread environmental pollution and enormous water consumption. Different units of a steel plant discharge effluents loaded with toxic, hazardous pollutants, and unutilized components which necessitates mitigation. In this paper, pollutant removal efficiency, effluent volume product quality, and economic feasibility of existing treatments are studied vis-à-vis their merits, demerits, and innovations to access their shortcomings which can be overcome with new technology to identify future research directions. While conventional methods are inadequate for complete remediation and water reclamation, the potential of advanced treatments, like membrane separation, remains relatively untapped. It is concluded that integrated systems combining membrane separation with chemical treatments can guarantee a high degree of contaminant removal, reusability of effluents concurrently leading to process intensification ensuring ecofriendliness and commercial viability.

The tethering of chromatin to the nuclear envelope supports nuclear mechanics

PubMed Central

Schreiner, Sarah M.; Koo, Peter K.; Zhao, Yao; Mochrie, Simon G. J.; King, Megan C.

2015-01-01

The nuclear lamina is thought to be the primary mechanical defence of the nucleus. However, the lamina is integrated within a network of lipids, proteins and chromatin; the interdependence of this network poses a challenge to defining the individual mechanical contributions of these components. Here, we isolate the role of chromatin in nuclear mechanics by using a system lacking lamins. Using novel imaging analyses, we observe that untethering chromatin from the inner nuclear membrane results in highly deformable nuclei in vivo, particularly in response to cytoskeletal forces. Using optical tweezers, we find that isolated nuclei lacking inner nuclear membrane tethers are less stiff than wild-type nuclei and exhibit increased chromatin flow, particularly in frequency ranges that recapitulate the kinetics of cytoskeletal dynamics. We suggest that modulating chromatin flow can define both transient and long-lived changes in nuclear shape that are biologically important and may be altered in disease. PMID:26074052

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

Tran, Ich C.; Tunuguntla, Ramya H.; Kim, Kyunghoon

Carbon nanotube porins (CNTPs), small segments of carbon nanotubes capable of forming defined pores in lipid membranes, are important future components for bionanoelectronic devices as they could provide a robust analog of biological membrane channels. Furthermore, in order to control the incorporation of these CNT channels into lipid bilayers, it is important to understand the structure of the CNTPs before and after insertion into the lipid bilayer as well as the impact of such insertion on the bilayer structure. Here we employed a noninvasive in situ probe, small-angle X-ray scattering, to study the integration of CNT porins into dioleoylphosphatidylcholine bilayers.more » These results show that CNTPs in solution are stabilized by a monolayer of lipid molecules wrapped around their outer surface. We also demonstrate that insertion of CNTPs into the lipid bilayer results in decreased bilayer thickness with the magnitude of this effect increasing with the concentration of CNTPs.« less

Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2.

PubMed

Wang, Jiarong; Li, Yan; Wang, Xiaoming; Chen, Wei; Sun, Hongbin; Wang, Junfeng

2014-11-01

Lipopolysaccharide (LPS), the important component of the outer membrane of Gram-negative bacteria, contributes to the integrity of the outer membrane and protects the cell against bactericidal agents, including antimicrobial peptides. However, the mechanisms of interaction between antimicrobial peptides and LPS are not clearly understood. Halictines-2 (HAL-2), one of the novel antimicrobial peptides, was isolated from the venom of the eusocial bee Halictus sexcinctus. HAL-2 has exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria and even against cancer cells. Here, we studied the interactions between HAL-2 and LPS to elucidate the antibacterial mechanism of HAL-2 in vitro. Our results show that HAL-2 adopts a significant degree of β-strand structure in the presence of LPS. LPS is capable of inducing HAL-2 amyloid formation, which may play a vital role in its antimicrobial activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterising the structural properties of polymer separators for lithium-ion batteries in 3D using phase contrast X-ray microscopy

NASA Astrophysics Data System (ADS)

Finegan, Donal P.; Cooper, Samuel J.; Tjaden, Bernhard; Taiwo, Oluwadamilola O.; Gelb, Jeff; Hinds, Gareth; Brett, Dan J. L.; Shearing, Paul R.

2016-11-01

Separators are an integral component for optimising performance and safety of lithium-ion batteries; therefore, a clear understanding of how their microstructure affects cell performance and safety is crucial. Phase contrast X-ray microscopy is used here to capture the microstructures of commercial monolayer, tri-layer, and ceramic-coated lithium-ion battery polymer separators. Spatial variations in key structural parameters, including porosity, tortuosity factor and pore size distribution, are determined through the application of 3D quantification techniques and stereology. The architectures of individual layers in multi-layer membranes are characterised, revealing anisotropy in porosity, tortuosity factor and mean pore size of the three types of separator. Detailed structural properties of the individual layers of multi-layered membranes are then related with their expected effect on safety and rate capability of cells.

Molecular physiology and modulation of somatodendritic A-type potassium channels.

PubMed

Jerng, Henry H; Pfaffinger, Paul J; Covarrubias, Manuel

2004-12-01

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.

Insight into mitochondrial structure and function from electron tomography.

PubMed

Frey, T G; Renken, C W; Perkins, G A

2002-09-10

In recent years, electron tomography has provided detailed three-dimensional models of mitochondria that have redefined our concept of mitochondrial structure. The models reveal an inner membrane consisting of two components, the inner boundary membrane (IBM) closely apposed to the outer membrane and the cristae membrane that projects into the matrix compartment. These two components are connected by tubular structures of relatively uniform size called crista junctions. The distribution of crista junction sizes and shapes is predicted by a thermodynamic model based upon the energy of membrane bending, but proteins likely also play a role in determining the conformation of the inner membrane. Results of structural studies of mitochondria during apoptosis demonstrate that cytochrome c is released without detectable disruption of the outer membrane or extensive swelling of the mitochondrial matrix, suggesting the formation of an outer membrane pore large enough to allow passage of holo-cytochrome c. The possible compartmentation of inner membrane function between the IBM and the cristae membrane is also discussed.

A mathematical model for predicting the life of polymer electrolyte fuel cell membranes subjected to hydration cycling

NASA Astrophysics Data System (ADS)

Burlatsky, S. F.; Gummalla, M.; O'Neill, J.; Atrazhev, V. V.; Varyukhin, A. N.; Dmitriev, D. V.; Erikhman, N. S.

2012-10-01

Under typical Polymer Electrolyte Membrane Fuel Cell (PEMFC) fuel cell operating conditions, part of the membrane electrode assembly is subjected to humidity cycling due to variation of inlet gas RH and/or flow rate. Cyclic membrane hydration/dehydration would cause cyclic swelling/shrinking of the unconstrained membrane. In a constrained membrane, it causes cyclic stress resulting in mechanical failure in the area adjacent to the gas inlet. A mathematical modeling framework for prediction of the lifetime of a PEMFC membrane subjected to hydration cycling is developed in this paper. The model predicts membrane lifetime as a function of RH cycling amplitude and membrane mechanical properties. The modeling framework consists of three model components: a fuel cell RH distribution model, a hydration/dehydration induced stress model that predicts stress distribution in the membrane, and a damage accrual model that predicts membrane lifetime. Short descriptions of the model components along with overall framework are presented in the paper. The model was used for lifetime prediction of a GORE-SELECT membrane.

Organic membrane photonic integrated circuits (OMPICs).

PubMed

Amemiya, Tomohiro; Kanazawa, Toru; Hiratani, Takuo; Inoue, Daisuke; Gu, Zhichen; Yamasaki, Satoshi; Urakami, Tatsuhiro; Arai, Shigehisa

2017-08-07

We propose the concept of organic membrane photonic integrated circuits (OMPICs), which incorporate various functions needed for optical signal processing into a flexible organic membrane. We describe the structure of several devices used within the proposed OMPICs (e.g., transmission lines, I/O couplers, phase shifters, photodetectors, modulators), and theoretically investigate their characteristics. We then present a method of fabricating the photonic devices monolithically in an organic membrane and demonstrate the operation of transmission lines and I/O couplers, the most basic elements of OMPICs.

The roles of bacteriophages in membrane-based water and wastewater treatment processes: A review.

PubMed

Wu, Bing; Wang, Rong; Fane, Anthony G

2017-03-01

Membrane filtration processes have been widely applied in water and wastewater treatment for many decades. Concerns related to membrane treatment effectiveness, membrane lifespan, and membrane fouling control have been paid great attention. To achieve sustainable membrane operation with regards to low energy and maintenance cost, monitoring membrane performance and applying suitable membrane control strategies are required. As the most abundant species in water and wastewater, bacteriophages have shown great potential to be employed in membrane processes as (1) indicators to assess membrane performance considering their similar properties to human pathogenic waterborne viruses; (2) surrogate particles to monitor membrane integrity due to their nano-sized nature; and (3) biological agents to alleviate membrane fouling because of their antimicrobial properties. This study aims to provide a comprehensive review on the roles of bacteriophages in membrane-based water and wastewater treatment processes, with focuses on their uses for membrane performance examination, membrane integrity monitoring, and membrane biofouling control. The advantages, limitations, and influencing factors for bacteriophage-based applications are reported. Finally, the challenges and prospects of bacteriophage-based applications in membrane processes for water treatment are highlighted. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lateral Organization of Lipids in Multi-component Liposomes

NASA Astrophysics Data System (ADS)

Ramachandran, Sanoop; Laradji, Mohamed; Sunil Kumar, P. B.

2009-04-01

Inspite of the fluid nature and low elastic modulus, membranes play a crucial role in maintaining the structural integrity of the cell. Recent experiments have challenged the passive nature of the membrane as proposed by the classical fluid mosaic model. Experiments indicate that biomembranes of eukaryotic cells may be laterally organized into small nanoscopic domains, called rafts, which are rich in sphingomyelin and cholesterol. It is largely believed that this in-plane organization is essential for a variety of physiological functions such as signaling, recruitment of specific proteins and endocytosis. However, elucidation of the fundamental issues including the mechanisms leading to the formation of lipid rafts, their stability, and their size remain difficult. This has reiterated the importance of understanding the equilibrium phase behavior and the kinetics of fluid multicomponent lipid membranes before attempts are made to find the effects of more complex mechanisms that may be involved in the formation and stability of lipid rafts. Current increase in interest in the domain formation in multicomponent membranes also stems from the experiments demonstrating fluid-fluid coexistence in mixtures of lipids and cholesterol and the success of several computational models in predicting their behavior. Here we review time dependent Ginzburg Landau model, dynamical triangulation Monte Carlo, and dissipative particle dynamics which are some of the methods that are commonly employed.

Robust membrane detection based on tensor voting for electron tomography.

PubMed

Martinez-Sanchez, Antonio; Garcia, Inmaculada; Asano, Shoh; Lucic, Vladan; Fernandez, Jose-Jesus

2014-04-01

Electron tomography enables three-dimensional (3D) visualization and analysis of the subcellular architecture at a resolution of a few nanometers. Segmentation of structural components present in 3D images (tomograms) is often necessary for their interpretation. However, it is severely hampered by a number of factors that are inherent to electron tomography (e.g. noise, low contrast, distortion). Thus, there is a need for new and improved computational methods to facilitate this challenging task. In this work, we present a new method for membrane segmentation that is based on anisotropic propagation of the local structural information using the tensor voting algorithm. The local structure at each voxel is then refined according to the information received from other voxels. Because voxels belonging to the same membrane have coherent structural information, the underlying global structure is strengthened. In this way, local information is easily integrated at a global scale to yield segmented structures. This method performs well under low signal-to-noise ratio typically found in tomograms of vitrified samples under cryo-tomography conditions and can bridge gaps present on membranes. The performance of the method is demonstrated by applications to tomograms of different biological samples and by quantitative comparison with standard template matching procedure. Copyright © 2014 Elsevier Inc. All rights reserved.

[Membrane model of the regulation of proliferation: the theory and interpretation of an experiment].

PubMed

Volkov, E I

1983-04-01

The role of cell surface physical organization in the cell cycle regulation is analyzed within the framework of the earlier proposed theory (Chernavskii et al., 1982). Two models of cell surface are considered: hard-frame fluid-mosaic model (latticemosaic) and the fluid-mosaic one. The former deals with normal cells. The existence of integral carcasse or "frame" which is formed by the essential part of cross-linked membrane components and may have at least two different conformational states is hypothesized. The second model describes membranes of tumour cells. With the latter theory any mitogen (excluding the restoration of nutrient depletion) reduces the mechanical tensile strength of the frame and stimulates the general structural rearrangement of the plasma membrane. There are only two conformational transitions during the cell cycle which serve as signals for the beginning of S and M phases. If the values of tensile strength are great enough and therefore the conformational transitions are impossible, the cells pass into the resting (prereplicative--G01, or premitotical--G02) state. Three types of experiments are interpreted in the proposed theory: a) on differences in the action of growth factors on normal and tumour cell cycle, b) on the necessary condition for mitogenicity of lectins, c) on the stimulation of proliferation by mechanical deformation of cells.

Proteoliposomes in nanobiotechnology.

PubMed

Ciancaglini, P; Simão, A M S; Bolean, M; Millán, J L; Rigos, C F; Yoneda, J S; Colhone, M C; Stabeli, R G

2012-03-01

Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multi-protein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

Modeling of membrane processes for air revitalization and water recovery

NASA Technical Reports Server (NTRS)

Lange, Kevin E.; Foerg, Sandra L.; Dall-Bauman, Liese A.

1992-01-01

Gas-separation and reverse-osmosis membrane models are being developed in conjunction with membrane testing at NASA JSC. The completed gas-separation membrane model extracts effective component permeabilities from multicomponent test data, and predicts the effects of flow configuration, operating conditions, and membrane dimensions on module performance. Variable feed- and permeate-side pressures are considered. The model has been applied to test data for hollow-fiber membrane modules with simulated cabin-air feeds. Results are presented for a membrane designed for air drying applications. Extracted permeabilities are used to predict the effect of operating conditions on water enrichment in the permeate. A first-order reverse-osmosis model has been applied to test data for spiral wound membrane modules with a simulated hygiene water feed. The model estimates an effective local component rejection coefficient under pseudosteady-state conditions. Results are used to define requirements for a detailed reverse-osmosis model.

Ultra-long-term cycling stability of an integrated carbon-sulfur membrane with dual shuttle-inhibiting layers of graphene "nets" and a porous carbon skin.

PubMed

Liu, Mingkai; Meng, Qinghua; Yang, Zhiyuan; Zhao, Xinsheng; Liu, Tianxi

2018-05-15

An integrated carbon-sulfur (CSG/PC) membrane with dual shuttle-inhibiting layers was prepared by inserting graphene "nets" and a porous carbon (PC) skin, and the membrane achieved an extraordinary cycling stability up to 1000 cycles with an average Coulombic efficiency of ∼100%.

Integration of lateral porous silicon membranes into planar microfluidics.

PubMed

Leïchlé, Thierry; Bourrier, David

2015-02-07

In this work, we present a novel fabrication process that enables the monolithic integration of lateral porous silicon membranes into single-layer planar microchannels. This fabrication technique relies on the patterning of local electrodes to guide pore formation horizontally within the membrane and on the use of silicon-on-insulator substrates to spatially localize porous silicon within the channel depth. The feasibility of our approach is studied by current flow analysis using the finite element method and supported by creating 10 μm long mesoporous membranes within 20 μm deep microchannels. The fabricated membranes are demonstrated to be potentially useful for dead-end microfiltration by adequately retaining 300 nm diameter beads while macromolecules such as single-stranded DNA and immunoglobulin G permeate the membrane. The experimentally determined fluidic resistance is in accordance with the theoretical value expected from the estimated pore size and porosity. The work presented here is expected to greatly simplify the integration of membranes capable of size exclusion based separation into fluidic devices and opens doors to the use of porous silicon in planar lab on a chip devices.

Using Haloarcula marismortui Bacteriorhodopsin as a Fusion Tag for Enhancing and Visible Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Hsu, Min-Feng; Yu, Tsung-Fu; Chou, Chia-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen; Wang, Andrew H. J.

2013-01-01

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system. PMID:23457558

Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes

USGS Publications Warehouse

Paniagua-Chavez, C. G.; Jenkins, J.; Segovia, M.; Tiersch, T.R.

2006-01-01

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P = 0.004) and thawing treatments (P = 0.04), and mitochondrial membrane potential (P = 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P = 0.0258) and with mitochondrial membrane potential (P = 0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P = 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced. ?? 2006 Elsevier Inc. All rights reserved.

Recent Advances in Pd-Based Membranes for Membrane Reactors.

PubMed

Arratibel Plazaola, Alba; Pacheco Tanaka, David Alfredo; Van Sint Annaland, Martin; Gallucci, Fausto

2017-01-01

Palladium-based membranes for hydrogen separation have been studied by several research groups during the last 40 years. Much effort has been dedicated to improving the hydrogen flux of these membranes employing different alloys, supports, deposition/production techniques, etc. High flux and cheap membranes, yet stable at different operating conditions are required for their exploitation at industrial scale. The integration of membranes in multifunctional reactors (membrane reactors) poses additional demands on the membranes as interactions at different levels between the catalyst and the membrane surface can occur. Particularly, when employing the membranes in fluidized bed reactors, the selective layer should be resistant to or protected against erosion. In this review we will also describe a novel kind of membranes, the pore-filled type membranes prepared by Pacheco Tanaka and coworkers that represent a possible solution to integrate thin selective membranes into membrane reactors while protecting the selective layer. This work is focused on recent advances on metallic supports, materials used as an intermetallic diffusion layer when metallic supports are used and the most recent advances on Pd-based composite membranes. Particular attention is paid to improvements on sulfur resistance of Pd based membranes, resistance to hydrogen embrittlement and stability at high temperature.

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

PubMed Central

van Geest, Marleen; Lolkema, Juke S.

2000-01-01

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies. PMID:10704472

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

PubMed Central

Wenzel, Michaela; Chiriac, Alina Iulia; Otto, Andreas; Zweytick, Dagmar; May, Caroline; Schumacher, Catherine; Gust, Ronald; Albada, H. Bauke; Penkova, Maya; Krämer, Ute; Erdmann, Ralf; Metzler-Nolte, Nils; Straus, Suzana K.; Bremer, Erhard; Becher, Dörte; Brötz-Oesterhelt, Heike; Sahl, Hans-Georg; Bandow, Julia Elisabeth

2014-01-01

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. PMID:24706874

Low-temperature bonded glass-membrane microfluidic device for in vitro organ-on-a-chip cell culture models

NASA Astrophysics Data System (ADS)

Pocock, Kyall J.; Gao, Xiaofang; Wang, Chenxi; Priest, Craig; Prestidge, Clive A.; Mawatari, Kazuma; Kitamori, Takehiko; Thierry, Benjamin

2015-12-01

The integration of microfluidics with living biological systems has paved the way to the exciting concept of "organson- a-chip", which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass based devices have long been utilised in the field of microfluidics but the integration of alternative functional elements within multi-layered glass microdevices, such as polymeric membranes, remains a challenge. To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimised on specialty low-temperature bonding equipment (μTAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units (EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650 °C) and quartz/fused silica bonding (1050 °C) processes, this method maintains the integrity and functionality of the membrane (Tg 150 °C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 hours, indicating sufficient bond strength for long term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days.

Low-temperature bonding process for the fabrication of hybrid glass-membrane organ-on-a-chip devices

NASA Astrophysics Data System (ADS)

Pocock, Kyall J.; Gao, Xiaofang; Wang, Chenxi; Priest, Craig; Prestidge, Clive A.; Mawatari, Kazuma; Kitamori, Takehiko; Thierry, Benjamin

2016-10-01

The integration of microfluidics with living biological systems has paved the way to the exciting concept of "organs-on-a-chip," which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass-based devices have long been utilized in the field of microfluidics but the integration of alternative functional elements within multilayered glass microdevices, such as polymeric membranes, remains a challenge. To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimized on specialty low-temperature bonding equipment (μTAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units (EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650°C) and quartz/fused silica bonding (1050°C) processes, this method maintains the integrity and functionality of the membrane (Tg 150°C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 h, indicating sufficient bond strength for long-term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days.

Perspective of Membrane Technology in Dairy Industry: A Review

PubMed Central

Kumar, Pavan; Sharma, Neelesh; Ranjan, Rajeev; Kumar, Sunil; Bhat, Z. F.; Jeong, Dong Kee

2013-01-01

Membrane technology has revolutionized the dairy sector. Different types of membranes are used in the industry for various purposes like extending the shelf life of milk without exposure to heat treatment, standardization of the major components of milk for tailoring new products as well increasing yield and quality of the dairy products, and concentrating, fractionation and purification of milk components especially valuable milk proteins in their natural state. In the cheese industry, membranes increase the yield and quality of cheese and control the whey volume, by concentrating the cheese milk. With the advancement of newer technology in membrane processes, it is possible to recover growth factor from whey. With the introduction of superior quality membranes as well as newer technology, the major limitation of membranes, fouling or blockage has been overcome to a greater extent. PMID:25049918

Perspective of membrane technology in dairy industry: a review.

PubMed

Kumar, Pavan; Sharma, Neelesh; Ranjan, Rajeev; Kumar, Sunil; Bhat, Z F; Jeong, Dong Kee

2013-09-01

Membrane technology has revolutionized the dairy sector. Different types of membranes are used in the industry for various purposes like extending the shelf life of milk without exposure to heat treatment, standardization of the major components of milk for tailoring new products as well increasing yield and quality of the dairy products, and concentrating, fractionation and purification of milk components especially valuable milk proteins in their natural state. In the cheese industry, membranes increase the yield and quality of cheese and control the whey volume, by concentrating the cheese milk. With the advancement of newer technology in membrane processes, it is possible to recover growth factor from whey. With the introduction of superior quality membranes as well as newer technology, the major limitation of membranes, fouling or blockage has been overcome to a greater extent.

Integration of cellular ubiquitin and membrane traffic systems: focus on deubiquitylases.

PubMed

Clague, Michael J; Urbé, Sylvie

2017-06-01

The cell is comprised of integrated multilevel protein networks or systems. The ubiquitin, protein homeostasis and membrane trafficking systems are highly integrated. Here, we look at the influence of reversible ubiquitylation on membrane trafficking and organelle dynamics. We review the regulation of endocytic sorting, selective autophagy and the secretory pathway by ubiquitin signals, with a particular focus on detailing the contribution of deubiquitylating enzymes. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Dehydration of an ethanol/water azeotrope by novel organic-inorganic hybrid membranes based on quaternized chitosan and tetraethoxysilane.

PubMed

Uragami, Tadashi; Katayama, Takuya; Miyata, Takashi; Tamura, Hiroshi; Shiraiwa, Tadashi; Higuchi, Akon

2004-01-01

To control swelling of quaternized chitosan (q-Chito) membranes, mixtures of q-Chito as an organic component and tetraethoxysilane (TEOS) as an inorganic component were prepared using the sol-gel reaction, and novel q-Chito/TEOS hybrid membranes were formed. In the separation of an ethanol/water azeotrope by pervaporation, the effect of TEOS content on the water/ethanol selectivity of q-Chito/TEOS hybrid membranes was investigated. Hybrid membranes containing up to 45 mol % TEOS exhibited higher water/ethanol selectivity than the q-Chito membrane. This resulted from depressed swelling of the membranes by formation of a cross-linked structure. However, introduction of excess TEOS led to greater swelling of the hybrid membranes. Therefore, the water/ethanol selectivity of the hybrid membranes containing more than 45 mol % TEOS was lower than that of the q-Chito membrane. The relationship between the structure of q-Chito/TEOS hybrid membranes and their permeation and separation characteristics during pervaporation of an ethanol/water azeotrope is discussed in detail.

Evaluation of amides and centrifugation temperature in boar semen cryopreservation.

PubMed

Bianchi, I; Calderam, K; Maschio, E F; Madeira, E M; da Rosa Ulguim, R; Corcini, C D; Bongalhardo, D C; Corrêa, E K; Lucia, T; Deschamps, J C; Corrêa, M N

2008-03-15

Two experiments were conducted to evaluate the use of amides as cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in boar semen cryopreservation protocols. Semen was diluted in BTS, cooled centrifuged, added to cooling extenders, followed by the addition of various cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5% dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%) were superior (P<0.05) to 5% methylformamide (MF; 43.2+/-2.4%) and 3% glycerol (GLY; 38.1+/-2.3%), with no significant difference between MF and GLY. Sperm membrane integrity was higher (P<0.05) for DMA than for MF or GLY (50.9+/-1.9, 43.3+/-2.5, and 34.5+/-2.8%, respectively). Sperm membrane integrity was higher in DMF (47.9+/-2.1%) than in glycerol (34.5+/-2.8%, P<0.05), but was similar to other treatments (P>0.05). In experiment 2, we tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and 47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P<0.05). For sperm motility and membrane integrity, 5% DMA exceeded (P<0.05) 3% DM, with greater membrane integrity than 3% DMF (P<0.05). In both experiments, sperm motility and membrane integrity were superior at 15 degrees C versus 24 degrees C (P<0.05), with no interaction between centrifugation temperature and treatments (P>0.05). In conclusion, boar semen was successfully cryopreserved by replacement of glycerol with amides (especially 5% DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm motility and membrane integrity.

Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions

PubMed Central

Vagne, Quentin; Turner, Matthew S.; Sens, Pierre

2015-01-01

The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal “corrals” and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles. PMID:26656912

Effect of pH buffer molecules on the light-induced currents from oriented purple membrane.

PubMed Central

Liu, S Y; Kono, M; Ebrey, T G

1991-01-01

The effect of pH buffers on the microsecond photocurrent component, B2, of oriented purple membranes has been studied. We found that under low salt conditions (less than 10 mM monovalent cationic salt) pH buffers can dramatically alter the waveform of the B2 component. The effect is induced by the protonation process of the buffer molecules by protons expelled from the membrane. These effects can be classified according to the charge transition upon protonation of the buffer. Buffers that carry two positive charges in their protonated form add a negative current component (N component) to B2. Almost all of the other buffers add a positive current component (P component) to B2, which is essentially a mirror image of the N component. Buffers with a pK less than 5.5 have only a small positive buffer component. The pH dependence of the buffer effect is closely related to the pK of the buffer; it requires that the buffer be in its unprotonated form. The rise time of the buffer component increases with the concentration of the buffer molecules. All the buffer effects can be inhibited by the addition of 5 mM of a divalent cation such as Ca2+. Reducing the surface potential slows down the N component but accelerates the P component without affecting the amplitude of the buffer effect significantly. Many of the buffer effects can be explained if we assume that upon protonation of the buffer by a proton expelled from the membrane by light, the buffer molecules move toward the membrane. This backward movement of buffer molecules forms a counter current very similar to that due to cations discussed in Liu, S. Y., R. Govindjee, and T. G. Ebrey. (1990. Biophys. J. 57:951-963). PMID:1883939

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

PubMed Central

Mitrea, Diana M; Cika, Jaclyn A; Guy, Clifford S; Ban, David; Banerjee, Priya R; Stanley, Christopher B; Nourse, Amanda; Deniz, Ashok A; Kriwacki, Richard W

2016-01-01

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus. DOI: http://dx.doi.org/10.7554/eLife.13571.001 PMID:26836305

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

DOE PAGES

Mitrea, Diana M.; Cika, Jaclyn A.; Guy, Clifford S.; ...

2016-02-02

In this study, the nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identifymore » multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.« less

Direct Measurement of Cyclic Current-Voltage Responses of Integral Membrane Proteins at a Self-Assembled Lipid-Bilayer-Modified Electrode: Cytochrome f and Cytochrome c Oxidase

NASA Astrophysics Data System (ADS)

Salamon, Z.; Hazzard, J. T.; Tollin, G.

1993-07-01

Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.

Ubiquitin-dependent sorting of integral membrane proteins for degradation in lysosomes

PubMed Central

Piper, Robert C.

2007-01-01

Summary The pathways that deliver newly synthesized proteins that reside in lysosomes are well understood by comparison with our knowledge of how integral membrane proteins are sorted and delivered to the lysosome for degradation. Many membrane proteins are sorted to lysosomes following ubiquitination, which provides a sorting signal that can operate for sorting at the TGN (trans-Golgi network), at the plasma membrane or at the endosome for delivery into lumenal vesicles. Candidate multicomponent machines that can potentially move ubiquitinated integral membrane cargo proteins have been identified, but much work is still required to ascertain which of these candidates directly recognizes ubiquitinated cargo and what they do with cargo after recognition. In the case of the machinery required for sorting into the lumenal vesicles of endosomes, other functions have also been determined including a link between sorting and movement of endosomes along microtubules. PMID:17689064

Effects of PEO-PPO-PEO Triblock Copolymers on Phospholipid Membrane Integrity under Osmotic Stress

PubMed Central

Wang, Jia-Yu; Chin, Jaemin; Marks, Jeremy D.; Lee, Ka Yee C.

2010-01-01

The effects of PEO-PPO-PEO triblock copolymers, mainly Poloxamer 188, on phospholipid membrane integrity under osmotic gradients were explored using giant unilamellar vesicles (GUVs). Fluorescence leakage assays showed two opposing effects of P188 on the structural integrity of GUVs depending on the duration of their incubation time. A two-state transition mechanism of interaction between the triblock copolymers and the phospholipid membrane is proposed: an adsorption (I) and an insertion (II) state. While the triblock copolymer in state I acts to moderately retard the leakage, their insertion in state II perturbs the lipid packing, thus increasing the membrane permeability. Our results suggest that the biomedical application of PEO-PPO-PEO triblock copolymers, either as cell membrane resealing agents or as accelerators for drug delivery, is directed by the delicate balance between these two states. PMID:20666423

Development of a preprototype thermoelectric integrated membrane evaporation subsystem for water recovery

NASA Technical Reports Server (NTRS)

Winkler, H. E.; Roebelen, G. J., Jr.

1980-01-01

A three-man urine water recovery preprototype subsystem using a new concept to provide efficient potable water recovery from waste fluids on extended duration space flights has been designed, fabricated, and tested. Low power, compactness, and gravity insensitive operation are featured in this vacuum distillation subsystem that combines a hollow fiber polysulfone membrane evaporator with a thermoelectric heat pump. Application and integration of these key elements have solved problems inherent in previous reclamation subsystem designs. The hollow fiber elements provide positive liquid/gas phase control with no moving parts other than a waste liquid recirculation pump and a product water withdrawal pump. Tubular membranes provide structural integrity, improving on previous flat sheet membrane designs. A thermoelectric heat pump provides latent energy recovery.

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton

PubMed Central

2017-01-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria. PMID:28945742

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

PubMed

Govindarajan, Sutharsan; Amster-Choder, Orna

2017-09-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

Impact of membrane curvature on amyloid aggregation.

PubMed

Terakawa, Mayu S; Lin, Yuxi; Kinoshita, Misaki; Kanemura, Shingo; Itoh, Dai; Sugiki, Toshihiko; Okumura, Masaki; Ramamoorthy, Ayyalusamy; Lee, Young-Ho

2018-04-28

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs. We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Recent Advances of Membrane-Cloaked Nanoplatforms for Biomedical Applications.

PubMed

Ai, Xiangzhao; Hu, Ming; Wang, Zhimin; Zhang, Wenmin; Li, Juan; Yang, Huanghao; Lin, Jun; Xing, Bengang

2018-04-18

In terms of the extremely small size and large specific surface area, nanomaterials often exhibit unusual physical and chemical properties, which have recently attracted considerable attention in bionanotechnology and nanomedicine. Currently, the extensive usage of nanotechnology in medicine holds great potential for precise diagnosis and effective therapeutics of various human diseases in clinical practice. However, a detailed understanding regarding how nanomedicine interacts with the intricate environment in complex living systems remains a pressing and challenging goal. Inspired by the diversified membrane structures and functions of natural prototypes, research activities on biomimetic and bioinspired membranes, especially for those cloaking nanosized platforms, have increased exponentially. By taking advantage of the flexible synthesis and multiple functionality of nanomaterials, a variety of unique nanostructures including inorganic nanocrystals and organic polymers have been widely devised to substantially integrate with intrinsic biomoieties such as lipids, glycans, and even cell and bacteria membrane components, which endow these abiotic nanomaterials with specific biological functionalities for the purpose of detailed investigation of the complicated interactions and activities of nanomedicine in living bodies, including their immune response activation, phagocytosis escape, and subsequent clearance from vascular system. In this review, we summarize the strategies established recently for the development of biomimetic membrane-cloaked nanoplatforms derived from inherent host cells (e.g., erythrocytes, leukocytes, platelets, and exosomes) and invasive pathogens (e.g., bacteria and viruses), mainly attributed to their versatile membrane properties in biological fluids. Meanwhile, the promising biomedical applications based on nanoplatforms inspired by diverse moieties, such as selective drug delivery in targeted sites and effective vaccine development for disease prevention, have also been outlined. Finally, the potential challenges and future prospects of the biomimetic membrane-cloaked nanoplatforms are also discussed.

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

PubMed

Muhammad, Saqib; Han, Shengli; Xie, Xiaoyu; Wang, Sicen; Aziz, Muhammad Majid

2017-01-01

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Irreversible, direct bonding of nanoporous polymer membranes to PDMS or glass microdevices.

PubMed

Aran, Kiana; Sasso, Lawrence A; Kamdar, Neal; Zahn, Jeffrey D

2010-03-07

A method for integrating porous polymer membranes such as polycarbonate, polyethersulfone and polyethylene terephthalate to microfluidic devices is described. The use of 3-aminopropyltriethoxysilane as a chemical crosslinking agent was extended to integrate membranes with PDMS and glass microfluidic channels. A strong, irreversible bond between the membranes and microfluidic structure was achieved. The bonding strength in the APTES treated devices was significantly greater than in devices fabricated using either a PDMS "glue" or two-part epoxy bonding method. Evaluation of a filtering microdevice and the pore structure via SEM indicates the APTES conjugation does not significantly alter the membrane transport function and pore morphology.

Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

PubMed Central

Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A.; Drosten, Christian; Thiel, Volker; Trybala, Edward

2014-01-01

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. PMID:24874215

Estradiol Membrane-Initiated Signaling and Female Reproduction.

PubMed

Micevych, Paul E; Wong, Angela May; Mittelman-Smith, Melinda Anne

2015-07-01

The discoveries of rapid, membrane-initiated steroid actions and central nervous system steroidogenesis have changed our understanding of the neuroendocrinology of reproduction. Classical nuclear actions of estradiol and progesterone steroids affecting transcription are essential. However, with the discoveries of membrane-associated steroid receptors, it is becoming clear that estradiol and progesterone have neurotransmitter-like actions activating intracellular events. Ultimately, membrane-initiated actions can influence transcription. Estradiol membrane-initiated signaling (EMS) modulates female sexual receptivity and estrogen feedback regulating the luteinizing hormone (LH) surge. In the arcuate nucleus, EMS activates a lordosis-regulating circuit that extends to the medial preoptic nucleus and subsequently to the ventromedial nucleus (VMH)--the output from the limbic and hypothalamic regions. Here, we discuss how EMS leads to an active inhibition of lordosis behavior. To stimulate ovulation, EMS facilitates astrocyte synthesis of progesterone (neuroP) in the hypothalamus. Regulation of GnRH release driving the LH surge is dependent on estradiol-sensitive kisspeptin (Kiss1) expression in the rostral periventricular nucleus of the third ventricle (RP3V). NeuroP activation of the LH surge depends on Kiss1, but the specifics of signaling have not been well elucidated. RP3V Kiss1 neurons appear to integrate estradiol and progesterone information which feeds back onto GnRH neurons to stimulate the LH surge. In a second population of Kiss1 neurons, estradiol suppresses the surge but maintains tonic LH release, another critical component of the estrous cycle. Together, evidence suggests that regulation of reproduction involves membrane action of steroids, some of which are synthesized in the brain. © 2015 American Physiological Society.

The amphiphilic alkyl ester derivatives of l-ascorbic acid induce reorganization of phospholipid vesicles.

PubMed

Giudice, Francesca; Ambroggio, Ernesto E; Mottola, Milagro; Fanani, Maria Laura

2016-09-01

l-ascorbic acid alkyl esters (ASCn) are lipophilic forms of vitamin C, which maintain some of its antioxidant power. Those properties make this drug family attractive to be used in pharmacological preparations protecting other redox-sensible drugs or designed to reduce possible toxic oxidative processes. In this work, we tested the ability of l-ascorbic acid alkyl esters (ASCn) to modulate the structure, permeability, and rheological properties of phospholipid bilayers. The ASCn studied here (ASC16, ASC14, and ASC12) alter the structural integrity as well as the rheological properties of phospholipid membranes without showing any evident detergent activity. ASC14 appeared as the most efficient drug in destabilize the membrane structure of nano- and micro-size phospholipid liposomes inducing vesicle content leakage and shape elongation on giant unilamellar vesicles. It also was the most potent enhancer of membrane microviscosity and surface water structuring. Only ASC16 induced the formation of drug-enriched condensed domains after its incorporation into the lipid bilayer, while ASC12 appeared as the less membrane-disturbing compound, likely because of its poor, and more superficial, partition into the membrane. We also found that incorporation of ASCn into the lipid bilayers enhanced the reduction of membrane components, compared with soluble vitamin C. Our study shows that ASCn compounds, which vary in the length of the acyl chain, show different effects on phospholipid vesicles used as biomembrane models. Those variances may account for subtly differences in the effectiveness on their pharmacological applications. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo voltage-dependent influences on summation of synaptic potentials in neurons of the lateral nucleus of the amygdala

PubMed Central

Rosenkranz, J. Amiel

2012-01-01

The amygdala has a fundamental role in driving affective behaviors in response to sensory cues. To accomplish this, neurons of the lateral nucleus (LAT) must integrate a large number of synaptic inputs. A wide range of factors influence synaptic integration, including membrane potential, voltage-gated ion channels and GABAergic inhibition. However, little is known about how these factors modulate integration of synaptic inputs in LAT neurons in vivo. The purpose of this study was to determine the voltage-dependent factors that modify in vivo integration of synaptic inputs in the soma of LAT neurons. In vivo intracellular recordings from anesthetized rats were used to measure post-synaptic potentials (PSPs) and clusters of PSPs across a range of membrane potentials. These studies found that the relationship between membrane potential and PSP clusters was sublinear, due to a reduction of cluster amplitude and area at depolarized membrane potentials. In combination with intracellular delivery of pharmacological agents, it was found that the voltage-dependent suppression of PSP clusters was sensitive to tetraethylammonium (TEA), but not cesium or a blocker of fast GABAergic inhibition. These findings indicate that integration of PSPs in LAT neurons in vivo is strongly modified by somatic membrane potential, likely through voltage-dependent TEA-sensitive potassium channels. Conditions that lead to a shift in membrane potential, or a modulation of the number or function of these ion channels will lead to a more uniform capacity for integration across voltages, and perhaps greatly facilitate amygdala-dependent behaviors. PMID:22989917

A novel approach for quantitative evaluation of the physicochemical interactions between rough membrane surface and sludge foulants in a submerged membrane bioreactor.

PubMed

Lin, Hongjun; Zhang, Meijia; Mei, Rongwu; Chen, Jianrong; Hong, Huachang

2014-11-01

This study proposed a novel approach for quantitative evaluation of the physicochemical interactions between a particle and rough surface. The approach adopts the composite Simpson's rule to numerically calculate the double integrals in the surface element integration of these physicochemical interactions. The calculation could be achieved by a MATLAB program based on this approach. This approach was then applied to assess the physicochemical interactions between rough membrane surface and sludge foulants in a submerged membrane bioreactor (MBR). The results showed that, as compared with smooth membrane surface, rough membrane surface had a much lower strength of interactions with sludge foulants. Meanwhile, membrane surface morphology significantly affected the strength and properties of the interactions. This study showed that the newly developed approach was feasible, and could serve as a primary tool for investigating membrane fouling in MBRs. Copyright © 2014 Elsevier Ltd. All rights reserved.

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

PubMed Central

Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

2008-01-01

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

PubMed

Arden, Catherine; Harbottle, Andrew; Baltrusch, Simone; Tiedge, Markus; Agius, Loranne

2004-09-01

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Polyacrylate microspheres composite for all-solid-state reference electrodes.

PubMed

Kisiel, Anna; Donten, Mikołaj; Mieczkowski, Józef; Rius-Ruiz, F Xavier; Maksymiuk, Krzysztof; Michalska, Agata

2010-09-01

A novel concept is proposed for the encapsulation of components within polyacrylate microspheres, prior to their incorporation into a membrane phase. Thus finer and better controlled dispersion of heterogeneous membrane components can be achieved. This concept was verified by using a poly(n-butyl acrylate) membrane-based reference electrode as an example. In this example the proper dispersion of solid constituents of the heterogeneous membrane and prevention of their leakage are both of primary importance. Potassium chloride-loaded poly(n-butyl acrylate) microspheres were prepared and then left in contact with silver nitrate to convert some of the KCl into AgCl. The material obtained was introduced into a poly(n-butyl acrylate) membrane. The reference electrode membranes obtained in this way were characterized with much more stable potential (both in different electrolytes and over time) compared with electrodes prepared by the direct introduction of KCl and AgCl to the membrane.

Two component-three dimensional catalysis

DOEpatents

Schwartz, Michael; White, James H.; Sammells, Anthony F.

2002-01-01

This invention relates to catalytic reactor membranes having a gas-impermeable membrane for transport of oxygen anions. The membrane has an oxidation surface and a reduction surface. The membrane is coated on its oxidation surface with an adherent catalyst layer and is optionally coated on its reduction surface with a catalyst that promotes reduction of an oxygen-containing species (e.g., O.sub.2, NO.sub.2, SO.sub.2, etc.) to generate oxygen anions on the membrane. The reactor has an oxidation zone and a reduction zone separated by the membrane. A component of an oxygen containing gas in the reduction zone is reduced at the membrane and a reduced species in a reactant gas in the oxidation zone of the reactor is oxidized. The reactor optionally contains a three-dimensional catalyst in the oxidation zone. The adherent catalyst layer and the three-dimensional catalyst are selected to promote a desired oxidation reaction, particularly a partial oxidation of a hydrocarbon.

Radiation effects on bovine taste bud membranes

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

Shatzman, A.R.; Mossman, K.L.

1982-11-01

In order to investigate the mechanisms of radiation-induced taste loss, the effects of radiation on preparations of enriched bovine taste bud membranes were studied. Taste buds containing circumvallate papilae, and surrounding control epithelial tissues devoid of taste buds, were obtained from steers and given radiation doses of 0-7000 cGy (rad). Tissue fractions were isolated into membrane-enriched and heterogeneous components using differential and sucrose gradient centrifugation of tissue homogenates. The yield of membranes, as measured by protein content in the buoyant membrane-enriched fractions, was reduced in quantity with increasing radiation dose. The relation between radiation dose and membrane quantity in membrane-enrichedmore » fractions could be fit by a simple exponential model with taste bud-derived membranes twice as radiosensitive as membranes from control epithelial tissue. Binding of sucrose, sodium, and acetate and fluoride stimulation of adenylate cyclase were nearly identical in both irradiated and nonirradiated intact membranes. Radiation had no effect on fractions of heterogeneous components. While it is not clear what changes are occurring in enriched taste cell membranes, damage to membranes may play an important role in the taste loss observed in patients following radiotherapy.« less

Hierarchical Inorganic Assemblies for Artificial Photosynthesis

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

Kim, Wooyul; Edri, Eran; Frei, Heinz

Artificial photosynthesis is an attractive approach for renewable fuel generation because it offers the prospect of a technology suitable for deployment on highly abundant, non-arable land. Recent leaps forward in the development of efficient and durable light absorbers and catalysts for oxygen evolution and the growing attention to catalysts for carbon dioxide activation brings into focus the tasks of hierarchically integrating the components into assemblies for closing of the photosynthetic cycle. A particular challenge is the efficient coupling of the multi-electron processes of CO 2 reduction and H 2O oxidation. Among the most important requirements for a complete integrated systemmore » are catalytic rates that match the solar flux, efficient charge transport between the various components, and scalability of the photosynthetic assembly on the unprecedented scale of terawatts in order to have impact on fuel consumption. To address these challenges, we have developed in this paper a heterogeneous inorganic materials approach with molecularly precise control of light absorption and charge transport pathways. Oxo-bridged heterobinuclear units with metal-to-metal charge-transfer transitions absorbing deep in the visible act as single photon, single charge transfer pumps for driving multi-electron catalysts. A photodeposition method has been introduced for the spatially directed assembly of nanoparticle catalysts for selective coupling to the donor or acceptor metal of the light absorber. For CO 2 reduction, a Cu oxide cluster is coupled to the Zr center of a ZrOCo light absorber, while coupling of an Ir nanoparticle catalyst for water oxidation to the Co donor affords closing of the photosynthetic cycle of CO 2 conversion by H 2O to CO and O 2. Optical, vibrational, and X-ray spectroscopy provide detailed structural knowledge of the polynuclear assemblies. Time resolved visible and rapid-scan FT-IR studies reveal charge transfer mechanisms and transient surface intermediates under photocatalytic conditions for guiding performance improvements. Separation of the water oxidation and carbon dioxide reduction half reactions by a membrane is essential for efficient photoreduction of CO 2 by H 2O to liquid fuel products. A concept of a macroscale artificial photosystem consisting of arrays of Co oxide–silica core–shell nanotubes is introduced in which each tube operates as a complete, independent photosynthetic unit with built-in membrane separation. The ultrathin amorphous silica shell with embedded molecular wires functions as a proton conducting, molecule impermeable membrane. Photoelectrochemical and transient optical measurements confirm tight control of charge transport through the membrane by the orbital energetics of the wire molecules. Finally, hierarchical arrangement of the components is accomplished by a combination of photodeposition, controlled anchoring, and atomic layer deposition methods.« less

Dipole potentials indicate restructuring of the membrane interface induced by gadolinium and beryllium ions

NASA Technical Reports Server (NTRS)

Ermakov, Y. A.; Averbakh, A. Z.; Yusipovich, A. I.; Sukharev, S.

2001-01-01

The dipole component of the membrane boundary potential, phi(d), is an integral parameter that may report on the conformational state of the lipid headgroups and their hydration. In this work, we describe an experimental approach to measurements of the dipole potential changes, Deltaphi(d), and apply it in studies of Be(2+) and Gd(3+) interactions with membranes composed of phosphatidylserine (PS), phosphatidylcholine (PC), and their mixtures. Deltaphi(d) is determined as the difference between the changes of the total boundary potential, phi(b), measured by the IFC method in planar lipid membranes and the surface potential, phi(s), determined from the electrophoretic mobility of liposomes. The Gouy-Chapman-Stern formalism, combined with the condition of mass balance, well describes the ion equilibria for these high-affinity cations. For the adsorption of Be(2+) and Gd(3+) to PC membranes, and of Mg(2+) to PS membranes, the values of Deltaphi(b) and Deltaphi(s) are the same, indicative of no change of phi(d). Binding of Gd(3+) to PS-containing membranes induces changes of phi(d) of opposite signs depending on the density of ionized PS headgroups in the bilayer. At low density, the induced Deltaphi(d) is negative (-30 mV), consistent with the effect of dehydration of the surface. At maximal density (pure PS, neutral pH), adsorption of Be(2+) or Gd(3+) induces an increase of phi(d) of 35 or 140 mV, respectively. The onset of the strong positive dipole effect on PS membranes with Gd(3+) is observed near the zero charge point and correlates with a six-fold increase of membrane tension. The observed phenomena may reflect concerted reorientation of dipole moments of PS headgroups as a result of ion adsorption and lipid condensation. Their possible implications to in-vivo effects of these high-affinity ions are discussed.

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations

PubMed Central

Fernandez, Fernando R.; Malerba, Paola; White, John A.

2015-01-01

The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na+ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances. PMID:25909971

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.

PubMed

Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Chen, Jianping

2014-06-01

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations.

PubMed

Fernandez, Fernando R; Malerba, Paola; White, John A

2015-04-01

The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na+ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances.

The retinal rod Na(+)/Ca(2+),K(+) exchanger contains a noncleaved signal sequence required for translocation of the N terminus.

PubMed

McKiernan, C J; Friedlander, M

1999-12-31

The retinal rod Na(+)/Ca(2+),K(+) exchanger (RodX) is a polytopic membrane protein found in photoreceptor outer segments where it is the principal extruder of Ca(2+) ions during light adaptation. We have examined the role of the N-terminal 65 amino acids in targeting, translocation, and integration of the RodX using an in vitro translation/translocation system. cDNAs encoding human RodX and bovine RodX through the first transmembrane domain were correctly targeted and integrated into microsomal membranes; deletion of the N-terminal 65 amino acids (aa) resulted in a translation product that was not targeted or integrated. Deletion of the first 65 aa had no effect on membrane targeting of full-length RodX, but the N-terminal hydrophilic domain no longer translocated. Chimeric constructs encoding the first 65 aa of bovine RodX fused to globin were translocated across microsomal membranes, demonstrating that the sequence could function heterologously. Studies of fresh bovine retinal extracts demonstrated that the first 65 aa are present in the native protein. These data demonstrate that the first 65 aa of RodX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of RodX.

Efficient ethanol recovery from fermentation broths with integrated distillation-membrane process

EPA Science Inventory

The energy demand of distillation-molecular sieve systems for ethanol recovery/dehydration can be significant, particularly for dilute solutions. An alternative process integrating vapor stripping (like a beer still) with vapor compression and a vapor permeation membrane separati...

Effect of storage in short--and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa.

PubMed

De Ambrogi, Marco; Ballester, Juan; Saravia, Fernando; Caballero, Ignacio; Johannisson, Anders; Wallgren, Margareta; Andersson, Magnus; Rodriguez-Martinez, Heriberto

2006-10-01

For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short--and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied.

Plasma membrane changes during the liquid storage of boar spermatozoa: a comparison of methods.

PubMed

Gaczarzewicz, Dariusz; Piasecka, Małgorzata; Udała, Jan; Błaszczyk, Barbara; Stankiewicz, Tomasz; Laszczyńska, Maria

2010-03-01

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 degrees C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of 'healthy' cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

The lysosomal membrane protein SCAV-3 maintains lysosome integrity and adult longevity

PubMed Central

Li, Yuan; Chen, Baohui; Zou, Wei; Wang, Xin; Wu, Yanwei; Zhao, Dongfeng; Sun, Yanan; Liu, Yubing

2016-01-01

Lysosomes degrade macromolecules and recycle metabolites as well as being involved in diverse processes that regulate cellular homeostasis. The lysosome is limited by a single phospholipid bilayer that forms a barrier to separate the potent luminal hydrolases from other cellular constituents, thus protecting the latter from unwanted degradation. The mechanisms that maintain lysosomal membrane integrity remain unknown. Here, we identified SCAV-3, the Caenorhabditis elegans homologue of human LIMP-2, as a key regulator of lysosome integrity, motility, and dynamics. Loss of scav-3 caused rupture of lysosome membranes and significantly shortened lifespan. Both of these phenotypes were suppressed by reinforced expression of LMP-1 or LMP-2, the C. elegans LAMPs, indicating that longevity requires maintenance of lysosome integrity. Remarkably, reduction in insulin/insulin-like growth factor 1 (IGF-1) signaling suppressed lysosomal damage and extended the lifespan in scav-3(lf) animals in a DAF-16–dependent manner. Our data reveal that SCAV-3 is essential for preserving lysosomal membrane stability and that modulation of lysosome integrity by the insulin/IGF-1 signaling pathway affects longevity. PMID:27810910

Integrated antibacterial and antifouling surfaces via cross-linking chitosan-g-eugenol/zwitterionic copolymer on electrospun membranes.

PubMed

Li, Zhenguang; Hu, Wenhong; Zhao, Yunhui; Ren, Lixia; Yuan, Xiaoyan

2018-04-27

Integrated antibacterial and antifouling surfaces in favor of avoiding implant-related infections are necessarily required for biomaterials when they contact with the body fluid. In this work, an antibacterial and antifouling membrane was developed via cross-linking chitosan-g-eugenol and the zwitterionic copolymer poly(sulfobetaine methylacrylate-co-2-aminoethyl methacrylate) on the electrospun polycarbonate urethane substrate using genipin as a cross-linker. Antibacterial assays demonstrated that the prepared membranes had efficient antibacterial activity with 92.8 ± 2.5% and 95.2 ± 1.3% growth inhibition rates against Escherichia coli and Staphylococcus aureus, respectively. The investigations on antifouling activity and hemocompatibility of the membranes showed significant resistances to bacterial attachment, non-specific protein adsorption and platelet adhesion, and presented lower hemolytic activity and good anticoagulant activity as well. Moreover, cell culture assays indicated that the prepared membranes exerted no obvious cytotoxicity with more than 80% of relative L929 fibroblast viability. Therefore, the membranes with integrated antibacterial and antifouling properties could be potentially applied in promising indwelling devices. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization and two-dimensional crystallization of membrane component AlkB of the medium-chain alkane hydroxylase system from Pseudomonas putida GPo1.

PubMed

Alonso, Hernan; Roujeinikova, Anna

2012-11-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C(12)E(8)]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-D-maltopyranoside (DM), n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.

Nuclear envelopathies: a complex LINC between nuclear envelope and pathology.

PubMed

Janin, Alexandre; Bauer, Delphine; Ratti, Francesca; Millat, Gilles; Méjat, Alexandre

2017-08-30

Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of "nuclear envelopathies" is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

A monolithic integrated micro direct methanol fuel cell based on sulfo functionalized porous silicon

NASA Astrophysics Data System (ADS)

Wang, M.; Lu, Y. X.; Liu, L. T.; Wang, X. H.

2016-11-01

In this paper, we demonstrate a monolithic integrated micro direct methanol fuel cell (μDMFC) for the first time. The monolithic integrated μDMFC combines proton exchange membrane (PEM) and Pt nanocatalysts, in which PEM is achieved by the functionalized porous silicon membrane and 3D Pt nanoflowers being synthesized in situ on it as catalysts. Sulfo groups functionalized porous silicon membrane serves as a PEM and a catalyst support simultaneously. The μDMFC prototype achieves an open circuit voltage of 0.3 V, a maximum power density of 5.5 mW/cm2. The monolithic integrated μDMFC offers several desirable features such as compatibility with micro fabrication techniques, an undeformable solid PEM and the convenience of assembly.

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

PubMed Central

Ichimura, Koichiro; Fukuyo, Yayoi; Nakamura, Tomomi; Powell, Rebecca; Sakai, Tatsuo; Janknecht, Ralf

2013-01-01

Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants. PMID:23324868

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

PubMed

Fujita, Miki; Lechner, Bettina; Barton, Deborah A; Overall, Robyn L; Wasteneys, Geoffrey O

2012-02-01

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.

Isolation of integrin-based adhesion complexes.

PubMed

Jones, Matthew C; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Robertson, Joseph; Paul, Nikki R; Ng, Daniel H J; Askari, Janet A; Humphries, Martin J

2015-03-02

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

MEMBRANE FILTER PROCEDURE FOR ENUMERATING THE COMPONENT GENERA OF THE COLIFORM GROUP IN SEAWATER

EPA Science Inventory

A facile, quantitative, membrane filter procedure (mC) for defining the distribution of coliform populations in seawater according to the component genera was developed. The procedure, which utilizes a series of in situ substrate tests to obviate the picking of colonies for ident...

Fabrication Methods and Performance of Low-Permeability Microfluidic Components for a Miniaturized Wearable Drug Delivery System

PubMed Central

Mescher, Mark J.; Swan, Erin E. Leary; Fiering, Jason; Holmboe, Maria E.; Sewell, William F.; Kujawa, Sharon G.; McKenna, Michael J.; Borenstein, Jeffrey T.

2010-01-01

In this paper, we describe low-permeability components of a microfluidic drug delivery system fabricated with versatile micromilling and lamination techniques. The fabrication process uses laminate sheets which are machined using XY milling tables commonly used in the printed-circuit industry. This adaptable platform for polymer microfluidics readily accommodates integration with silicon-based sensors, printed-circuit, and surface-mount technologies. We have used these methods to build components used in a wearable liquid-drug delivery system for in vivo studies. The design, fabrication, and performance of membrane-based fluidic capacitors and manual screw valves provide detailed examples of the capability and limitations of the fabrication method. We demonstrate fluidic capacitances ranging from 0.015 to 0.15 μL/kPa, screw valves with on/off flow ratios greater than 38 000, and a 45× reduction in the aqueous fluid loss rate to the ambient due to permeation through a silicone diaphragm layer. PMID:20852729

Partitioning behavior of aromatic components in jet fuel into diverse membrane-coated fibers.

PubMed

Baynes, Ronald E; Xia, Xin-Rui; Barlow, Beth M; Riviere, Jim E

2007-11-01

Jet fuel components are known to partition into skin and produce occupational irritant contact dermatitis (OICD) and potentially adverse systemic effects. The purpose of this study was to determine how jet fuel components partition (1) from solvent mixtures into diverse membrane-coated fibers (MCFs) and (2) from biological media into MCFs to predict tissue distribution. Three diverse MCFs, polydimethylsiloxane (PDMS, lipophilic), polyacrylate (PA, polarizable), and carbowax (CAR, polar), were selected to simulate the physicochemical properties of skin in vivo. Following an appropriate equilibrium time between the MCF and dosing solutions, the MCF was injected directly into a gas chromatograph/mass spectrometer (GC-MS) to quantify the amount that partitioned into the membrane. Three vehicles (water, 50% ethanol-water, and albumin-containing media solution) were studied for selected jet fuel components. The more hydrophobic the component, the greater was the partitioning into the membranes across all MCF types, especially from water. The presence of ethanol as a surrogate solvent resulted in significantly reduced partitioning into the MCFs with discernible differences across the three fibers based on their chemistries. The presence of a plasma substitute (media) also reduced partitioning into the MCF, with the CAR MCF system being better correlated to the predicted partitioning of aromatic components into skin. This study demonstrated that a single or multiple set of MCF fibers may be used as a surrogate for octanol/water systems and skin to assess partitioning behavior of nine aromatic components frequently formulated with jet fuels. These diverse inert fibers were able to assess solute partitioning from a blood substitute such as media into a membrane possessing physicochemical properties similar to human skin. This information may be incorporated into physiologically based pharmacokinetic (PBPK) models to provide a more accurate assessment of tissue dosimetry of related toxicants.

Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.

PubMed Central

Pakrasi, H B; De Ciechi, P; Whitmarsh, J

1991-01-01

Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII. Images PMID:1904816

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age

PubMed Central

Oiki, Shigetoshi

2015-01-01

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the ‘frozen’ crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. PMID:25833254

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

PubMed

Oiki, Shigetoshi

2015-06-15

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Polymeric and Lipid Membranes-From Spheres to Flat Membranes and vice versa.

PubMed

Saveleva, Mariia S; Lengert, Ekaterina V; Gorin, Dmitry A; Parakhonskiy, Bogdan V; Skirtach, Andre G

2017-08-15

Membranes are important components in a number of systems, where separation and control of the flow of molecules is desirable. Controllable membranes represent an even more coveted and desirable entity and their development is considered to be the next step of development. Typically, membranes are considered on flat surfaces, but spherical capsules possess a perfect "infinite" or fully suspended membranes. Similarities and transitions between spherical and flat membranes are discussed, while applications of membranes are also emphasized.

Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway

PubMed Central

Brigé, Ann; Motte, Bart; Borloo, Jimmy; Buysschaert, Géraldine; Devreese, Bart; Van Beeumen, Jozef J.

2008-01-01

Summary Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye‐containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR‐1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox‐active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin. PMID:21261820

Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

PubMed

Jouhet, Juliette; Gray, John C

2009-10-01

Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

A biofilter integrated with gas membrane separation unit for the treatment of fluctuating styrene loads.

PubMed

Li, Lin; Lian, Jing; Han, Yunping; Liu, Junxin

2012-05-01

Biofiltration for volatile organic compound control in waste gas streams is best operated at steady contaminant loadings. To provide long-term stable operation of a biofilter under adverse contaminant feeding conditions, an integrated bioreactor system with a gas separation membrane module installed after a biofilter was proposed for styrene treatment. Styrene was treated effectively, with average styrene effluent concentrations maintained at less than 50 mg m(-3) and a total removal efficiency of over 96% achieved when the biofiltration column faced fluctuating loads. The maximum elimination capacity of the integrated bioreactor system was 93.8 g m(-3)h(-1), which was higher than that obtained with the biofiltration column alone. The combination of these two processes (microbial and chemical) led to more efficient elimination of styrene and buffering of the fluctuating loads. The factors on gas membrane separation, microbial characteristics in the integrated bioreactor and membrane fouling were also investigated in this study. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ethanol fermentation integrated with PDMS composite membrane: An effective process.

PubMed

Fu, Chaohui; Cai, Di; Hu, Song; Miao, Qi; Wang, Yong; Qin, Peiyong; Wang, Zheng; Tan, Tianwei

2016-01-01

The polydimethylsiloxane (PDMS) membrane, prepared in water phase, was investigated in separation ethanol from model ethanol/water mixture and fermentation-pervaporation integrated process. Results showed that the PDMS membrane could effectively separate ethanol from model solution. When integrated with batch ethanol fermentation, the ethanol productivity was enhanced compared with conventional process. Fed-batch and continuous ethanol fermentation with pervaporation were also performed and studied. 396.2-663.7g/m(2)h and 332.4-548.1g/m(2)h of total flux with separation factor of 8.6-11.7 and 8-11.6, were generated in the fed-batch and continuous fermentation with pervaporation scenario, respectively. At the same time, high titre ethanol production of ∼417.2g/L and ∼446.3g/L were also achieved on the permeate side of membrane in the two scenarios, respectively. The integrated process was environmental friendly and energy saving, and has a promising perspective in long-terms operation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fabrication of a Cryogenic Terahertz Emitter for Bolometer Focal Plane Calibrations

NASA Technical Reports Server (NTRS)

Chervenak, James; Brown, Ari; Wollack, Edward

2012-01-01

A fabrication process is reported for prototype emitters of THz radiation, which operate cryogenically, and should provide a fast, stable blackbody source suitable for characterization of THz devices. The fabrication has been demonstrated and, at the time of this reporting, testing was underway. The emitter is similar to a monolithic silicon bolometer in design, using both a low-noise thermometer and a heater element on a thermally isolated stage. An impedance-matched, high-emissivity coat ing is also integrated to tune the blackbody properties. This emitter is designed to emit a precise amount of power as a blackbody spectrum centered on terahertz frequencies. The emission is a function of the blackbody temperature. An integrated resistive heater and thermometer system can control the temperature of the blackbody with greater precision than previous incarnations of calibration sources that relied on blackbody emission. The emitter is fabricated using a silicon- on-insulator substrate wafer. The buried oxide is chosen to be less than 1 micron thick, and the silicon device thickness is 1-2 microns. Layers of phosphorus compensated with boron are implanted into and diffused throughout the full thickness of the silicon device layer to create the thermometer and heater components. Degenerately doped wiring is implanted to connect the devices to wire-bondable contact pads at the edge of the emitter chip. Then the device is micromachined to remove the thick-handle silicon behind the thermometer and heater components, and to thermally isolate it on a silicon membrane. An impedance- matched emissive coating (ion assisted evaporated Bi) is applied to the back of the membrane to enable high-efficiency emission of the blackbody spectrum.

Direct ultrafiltration performance and membrane integrity monitoring by microbiological analysis.

PubMed

Ferrer, O; Casas, S; Galvañ, C; Lucena, F; Bosch, A; Galofré, B; Mesa, J; Jofre, J; Bernat, X

2015-10-15

The feasibility of substituting a conventional pre-treatment, consisting of dioxi-chlorination, coagulation/flocculation, settling and sand filtration, of a drinking water treatment plant (DWTP) by direct ultrafiltration (UF) has been assessed from a microbiological standpoint. Bacterial indicators, viral indicators and human viruses have been monitored in raw river, ultrafiltered and conventionally pre-treated water samples during two years. Direct UF has proven to remove bacterial indicators quite efficiently and to a greater extent than the conventional process does. Nevertheless, the removal of small viruses such as some small bacteriophages and human viruses (e.g. enteroviruses and noroviruses) is lower than the current conventional pre-treatment. Membrane integrity has been assessed during two years by means of tailored tests based on bacteriophages with different properties (MS-2, GA and PDR-1) and bacterial spores (Bacillus spores). Membrane integrity has not been compromised despite the challenging conditions faced by directly treating raw river water. Bacteriophage PDR-1 appears as a suitable microbe to test membrane integrity, as its size is slightly larger than the considered membrane pore size. However, its implementation at full scale plant is still challenging due to difficulties in obtaining enough phages for its seeding. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Critical time window of neuronal cholesterol synthesis during neurite outgrowth.

PubMed

Fünfschilling, Ursula; Jockusch, Wolf J; Sivakumar, Nandhini; Möbius, Wiebke; Corthals, Kristina; Li, Sai; Quintes, Susanne; Kim, Younghoon; Schaap, Iwan A T; Rhee, Jeong-Seop; Nave, Klaus-Armin; Saher, Gesine

2012-05-30

Cholesterol is an essential membrane component enriched in plasma membranes, growth cones, and synapses. The brain normally synthesizes all cholesterol locally, but the contribution of individual cell types to brain cholesterol metabolism is unknown. To investigate whether cortical projection neurons in vivo essentially require cholesterol biosynthesis and which cell types support neurons, we have conditionally ablated the cholesterol biosynthesis in these neurons in mice either embryonically or postnatally. We found that cortical projection neurons synthesize cholesterol during their entire lifetime. At all stages, they can also benefit from glial support. Adult neurons that lack cholesterol biosynthesis are mainly supported by astrocytes such that their functional integrity is preserved. In contrast, microglial cells support young neurons. However, compensatory efforts of microglia are only transient leading to layer-specific neuronal death and the reduction of cortical projections. Hence, during the phase of maximal membrane growth and maximal cholesterol demand, neuronal cholesterol biosynthesis is indispensable. Analysis of primary neurons revealed that neurons tolerate only slight alteration in the cholesterol content and plasma membrane tension. This quality control allows neurons to differentiate normally and adjusts the extent of neurite outgrowth, the number of functional growth cones and synapses to the available cholesterol. This study highlights both the flexibility and the limits of horizontal cholesterol transfer in vivo and may have implications for the understanding of neurodegenerative diseases.

Electron transport and light-harvesting switches in cyanobacteria

PubMed Central

Mullineaux, Conrad W.

2014-01-01

Cyanobacteria possess multiple mechanisms for regulating the pathways of photosynthetic and respiratory electron transport. Electron transport may be regulated indirectly by controlling the transfer of excitation energy from the light-harvesting complexes, or it may be more directly regulated by controlling the stoichiometry, localization, and interactions of photosynthetic and respiratory electron transport complexes. Regulation of the extent of linear vs. cyclic electron transport is particularly important for controlling the redox balance of the cell. This review discusses what is known of the regulatory mechanisms and the timescales on which they occur, with particular regard to the structural reorganization needed and the constraints imposed by the limited mobility of membrane-integral proteins in the crowded thylakoid membrane. Switching mechanisms requiring substantial movement of integral thylakoid membrane proteins occur on slower timescales than those that require the movement only of cytoplasmic or extrinsic membrane proteins. This difference is probably due to the restricted diffusion of membrane-integral proteins. Multiple switching mechanisms may be needed to regulate electron transport on different timescales. PMID:24478787

Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Ungar, Eugene K.; Almlie, Jay C.

2010-01-01

A water membrane evaporator (WME) has been conceived and tested as an alternative to the contamination-sensitive and corrosion-prone evaporators currently used for dissipating heat from space vehicles. The WME consists mainly of the following components: An outer stainless-steel screen that provides structural support for the components mentioned next; Inside and in contact with the stainless-steel screen, a hydrophobic membrane that is permeable to water vapor; Inside and in contact with the hydrophobic membrane, a hydrophilic membrane that transports the liquid feedwater to the inner surface of the hydrophobic membrane; Inside and in contact with the hydrophilic membrane, an annular array of tubes through which flows the spacecraft coolant carrying the heat to be dissipated; and An inner exclusion tube that limits the volume of feedwater in the WME. In operation, a pressurized feedwater reservoir is connected to the volume between the exclusion tube and the coolant tubes. Feedwater fills the volume, saturates the hydrophilic membrane, and is retained by the hydrophobic membrane. The outside of the WME is exposed to space vacuum. Heat from the spacecraft coolant is conducted through the tube walls and the water-saturated hydrophilic membrane to the liquid/vapor interface at the hydrophobic membrane, causing water to evaporate to space. Makeup water flows into the hydrophilic membrane through gaps between the coolant tubes.

Transferrable monolithic III-nitride photonic circuit for multifunctional optoelectronics

NASA Astrophysics Data System (ADS)

Shi, Zheng; Gao, Xumin; Yuan, Jialei; Zhang, Shuai; Jiang, Yan; Zhang, Fenghua; Jiang, Yuan; Zhu, Hongbo; Wang, Yongjin

2017-12-01

A monolithic III-nitride photonic circuit with integrated functionalities was implemented by integrating multiple components with different functions into a single chip. In particular, the III-nitride-on-silicon platform is used as it integrates a transmitter, a waveguide, and a receiver into a suspended III-nitride membrane via a wafer-level procedure. Here, a 0.8-mm-diameter suspended device architecture is directly transferred from silicon to a foreign substrate by mechanically breaking the support beams. The transferred InGaN/GaN multiple-quantum-well diode (MQW-diode) exhibits a turn-on voltage of 2.8 V with a dominant electroluminescence peak at 453 nm. The transmitter and receiver share an identical InGaN/GaN MQW structure, and the integrated photonic circuit inherently works for on-chip power monitoring and in-plane visible light communication. The wire-bonded monolithic photonic circuit on glass experimentally demonstrates in-plane data transmission at 120 Mb/s, paving the way for diverse applications in intelligent displays, in-plane light communication, flexible optical sensors, and wearable III-nitride optoelectronics.

Superior Robust Ultrathin Single-Crystalline Silicon Carbide Membrane as a Versatile Platform for Biological Applications.

PubMed

Nguyen, Tuan-Khoa; Phan, Hoang-Phuong; Kamble, Harshad; Vadivelu, Raja; Dinh, Toan; Iacopi, Alan; Walker, Glenn; Hold, Leonie; Nguyen, Nam-Trung; Dao, Dzung Viet

2017-12-06

Micromachined membranes are promising platforms for cell culture thanks to their miniaturization and integration capabilities. Possessing chemical inertness, biocompatibility, and integration, silicon carbide (SiC) membranes have attracted great interest toward biological applications. In this paper, we present the batch fabrication, mechanical characterizations, and cell culture demonstration of robust ultrathin epitaxial deposited SiC membranes. The as-fabricated ultrathin SiC membranes, with an ultrahigh aspect ratio (length/thickness) of up to 20 000, possess high a fracture strength up to 2.95 GPa and deformation up to 50 μm. A high optical transmittance of above 80% at visible wavelengths was obtained for 50 nm membranes. The as-fabricated membranes were experimentally demonstrated as an excellent substrate platform for bio-MEMS/NEMS cell culture with the cell viability rate of more than 92% after 72 h. The ultrathin SiC membrane is promising for in vitro observations/imaging of bio-objects with an extremely short optical access.

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

PubMed Central

Hoogerheide, David P.; Noskov, Sergei Y.; Jacobs, Daniel; Bergdoll, Lucie; Silin, Vitalii; Worcester, David L.; Abramson, Jeff; Nanda, Hirsh; Rostovtseva, Tatiana K.; Bezrukov, Sergey M.

2017-01-01

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents. PMID:28420794

The connection of cytoskeletal network with plasma membrane and the cell wall

PubMed Central

Liu, Zengyu; Persson, Staffan; Zhang, Yi

2015-01-01

The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field. PMID:25693826

Antimetastatic effect of PSK, a protein-bound polysaccharide, against the B16-BL6 mouse melanoma.

PubMed

Matsunaga, K; Ohhara, M; Oguchi, Y; Iijima, H; Kobayashi, H

1996-01-01

We examined the effect of PSK, a protein-bound polysaccharide, upon in vivo metastasis and in vitro invasion of the B16-BL6 mouse melanoma cells. (1) PSK suppressed in vivo artificial and spontaneous lung metastases of B16-BL6 in C57BL/6 mice. (2) PSK in a dose-dependent fashion suppressed in vitro invasion and chemotaxis of the tumor cells using filters coated with a reconstituted basement membrane. (3) PSK had little effect on DNA synthesis in tumor cells in vitro, but suppressed tumor cell adhesion to, degradation of, and haptotaxis to components of the basement membrane. (4) PSK suppressed the binding of tumor cells to components of the basement membrane. These findings suggest that PSK may suppress metastasis through inhibition of tumor cell invasion and that this effect is the result of interactions between PSK and components of the basement membrane.

Modeling the light-induced electric potential difference (ΔΨ), the pH difference (ΔpH) and the proton motive force across the thylakoid membrane in C3 leaves.

PubMed

Lyu, Hui; Lazár, Dušan

2017-01-21

A model was constructed which includes electron transport (linear and cyclic and Mehler type reaction) coupled to proton translocation, counter ion movement, ATP synthesis, and Calvin-Benson cycle. The focus is on modeling of the light-induced total electric potential difference (ΔΨ) which in this model originates from the bulk phase electric potential difference (ΔΨ b ), the localized electric potential difference (ΔΨ c ), as well as the surface electric potential difference (ΔΨ s ). The measured dual wavelength transmittance signal (ΔA515-560nm, electrochromic shift) was used as a proxy for experimental ΔΨ. The predictions for theoretical ΔΨ vary with assumed contribution of ΔΨ s , which might imply that the measured ΔA515-560nm trace on a long time scale reflects the interplay of the ΔΨ components. Simulations also show that partitioning of proton motive force (pmf) to ΔΨ b and ΔpH components is sensitive to the stoichiometric ratio of H + /ATP, energy barrier for ATP synthesis, ionic strength, buffer capacity and light intensity. Our model shows that high buffer capacity promotes the establishment of ΔΨ b , while the formation of pH i minimum is not 'dissipated' but 'postponed' until it reaches the same level as that for low buffer capacity. Under physiologically optimal conditions, the output of the model shows that at steady state in light, the ΔpH component is the main contributor to pmf to drive ATP synthesis while a low ΔΨ b persists energizing the membrane. Our model predicts 11mV as the resting electric potential difference across the thylakoid membrane in dark. We suggest that the model presented in this work can be integrated as a module into a more comprehensive model of oxygenic photosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mucins in contact lens wear and dry eye conditions.

PubMed

Ramamoorthy, Padmapriya; Nichols, Jason J

2008-08-01

Ocular mucins are thought to play integral roles in ocular surface lubrication, anchoring of the aqueous, stabilizing the lipid components of the tear film, eliminating foreign bodies and pathogens, and with potential involvement in cell cycle mediation and apoptotic activity of ocular surface epithelia. Ocular mucins are of secreted and membrane-associated types. Secreted mucins may be of large gel-forming type or small soluble mucins (e.g., MUC5AC and MUC7). Membrane-associated mucins such as MUCs 1 and 4 are a major component of the glycocalyx. They are thought to render structural support to the microplicae and mediate epithelial cell cycle and apoptotic activity. The alterations in ocular mucins with contact lens wear are unclear. Recent work shows mucin expression may be up-regulated during the early years of contact lens wear, and with long-term lens wear, mucin expression may return to normal levels or sub-normal levels, although this is not well understood. Further, the polar nature of mucins may be associated with their affinity for contact lens surfaces making them a component of contact lens deposition. This has potential implications in the wettability and tolerability of contact lenses, and may be impacted by surface coatings, polymer characteristics, or care solutions. Conjunctival mucin gene expression and secretion may be deficient in several ocular surface disorders associated with dry eye. Deficiency and alterations in glycosylation characteristics of MUC5AC and MUC2 have been reported in both Sjögren and non-Sjögren dry eye types. Decreased binding of the membrane-associated mucin MUC16 to the conjunctival epithelium has been reported in Sjögren dry eye while MUC1 alterations have been reported in Sjögren and non-Sjögren dry eye states. In view of the mucin involvement in dry eye conditions, stimulation of mucus secretion pathways may hold promise in the pharmaceutical treatment of dry eye.

Cytoskeletal Components Define Protein Location to Membrane Microdomains*

PubMed Central

Szymanski, Witold G.; Zauber, Henrik; Erban, Alexander; Gorka, Michal; Wu, Xu Na; Schulze, Waltraud X.

2015-01-01

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. PMID:26091700

INTEGRATION OF FILTRATION AND ADVANCED OXIDATION: DEVELOPMENT OF A MEMBRANE LIQUID-PHASE PLASMA REACTOR

EPA Science Inventory

A tiered approach will be undertaken to achieve the overall project goal of demonstrating the integrated membrane/plasma process as an innovative, affordable, sustainable and effective treatment technology for small treatment systems. The team will first use a regimented ap...

Risk assessment of Giardia from a full scale MBR sewage treatment plant caused by membrane integrity failure.

PubMed

Zhang, Yu; Chen, Zhimin; An, Wei; Xiao, Shumin; Yuan, Hongying; Zhang, Dongqing; Yang, Min

2015-04-01

Membrane bioreactors (MBR) are highly efficient at intercepting particles and microbes and have become an important technology for wastewater reclamation. However, many pathogens can accumulate in activated sludge due to the long residence time usually adopted in MBR, and thus may pose health risks when membrane integrity problems occur. This study presents data from a survey on the occurrence of water-borne Giardia pathogens in reclaimed water from a full-scale wastewater treatment plant with MBR experiencing membrane integrity failure, and assessed the associated risk for green space irrigation. Due to membrane integrity failure, the MBR effluent turbidity varied between 0.23 and 1.90 NTU over a period of eight months. Though this turbidity level still met reclaimed water quality standards (≤5 NTU), Giardia were detected at concentrations of 0.3 to 95 cysts/10 L, with a close correlation between effluent turbidity and Giardia concentration. All β-giardin gene sequences of Giardia in the WWTP influents were genotyped as Assemblages A and B, both of which are known to infect humans. An exponential dose-response model was applied to assess the risk of infection by Giardia. The risk in the MBR effluent with chlorination was 9.83×10(-3), higher than the acceptable annual risk of 1.0×10(-4). This study suggested that membrane integrity is very important for keeping a low pathogen level, and multiple barriers are needed to ensure the biological safety of MBR effluent. Copyright © 2015. Published by Elsevier B.V.

An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

PubMed

Ihekwaba, Adaoha E C; Mura, Ivan; Walshaw, John; Peck, Michael W; Barker, Gary C

2016-11-01

Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes

PubMed Central

Goren, Michael A.; Nozawa, Akira; Makino, Shin-ichi; Wrobel, Russell L.; Fox, Brian G.

2018-01-01

Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies. PMID:19892197

[Effect of damage integrity rat brain synaptic membranes on the functional activity GABA(A)-receptor/Cl(-)-ionophore complex in the CNC].

PubMed

Rebrov, I G; Kalinina, M V

2013-01-01

Functional activity of the CGABA(A)-receptor/Cl(-) ionophore complex was investigated the muscimol-stimulated entry of the radioactive isotope 36Cl(-) in synaptoneurosomes in changing the structure and permeability of neuronal membranes. Integrity of the membranes was damaged by removal of Ca(+2) and Mg(+2) from the incubation medium and by the method of freezing-thawing synaptoneurosomes. In both cases, an increase in basal 36Cl(-) entry into synaptoneurosomes, indicating increased nonspecific permeability of neuronal membranes, and decreased activity the CABA(A)-receptor/Cl(-) ionophore complex. The conclusion about the relationship of processes damage neuronal membranes and reducing the inhibitory processes in the epileptic focus.

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

PubMed Central

Öjemalm, Karin; Halling, Katrin K.; Nilsson, IngMarie; von Heijne, Gunnar

2013-01-01

Summary α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α-helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α-helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (Ncyt-Clum or Nlum-Ccyt) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the ‘missing hydrophobicity’ in polytopic membrane proteins. PMID:22281052

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli.

PubMed

Hou, Xiaofang; Wang, Sicen; Hou, Jingjing; He, Langchong

2011-03-01

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

PubMed

Kreidler, Anna-Maria; Benz, Roland; Barth, Holger

2017-03-01

The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B 7 ) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B 7 components of C2 toxin and LT, C2IIa and PA 63 , respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA 63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA 63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA 63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA 63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

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

PubMed Central

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

2014-01-01

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

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

DOE PAGES

Seppala, Susanna; Solomon, Kevin V.; Gilmore, Sean P.; ...

2016-12-20

Here, engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive andmore » rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. In conclusion, we report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.« less

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

NASA Technical Reports Server (NTRS)

Costes, S.; Streuli, C. H.; Barcellos-Hoff, M. H.

2000-01-01

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

Proteomic Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins*

PubMed Central

Clarke, Victoria C.; Loughlin, Patrick C.; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M.; Day, David A.; Smith, Penelope M.C.

2015-01-01

Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID:25724908

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters.

PubMed

Seppälä, Susanna; Solomon, Kevin V; Gilmore, Sean P; Henske, John K; O'Malley, Michelle A

2016-12-20

Engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. We report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.

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

PubMed

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

2007-12-28

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

Primary cellular meningeal defects cause neocortical dysplasia and dyslamination

PubMed Central

Hecht, Jonathan H.; Siegenthaler, Julie A.; Patterson, Katelin P.; Pleasure, Samuel J.

2010-01-01

Objective Cortical malformations are important causes of neurological morbidity, but in many cases their etiology is poorly understood. Mice with Foxc1 mutations have cellular defects in meningeal development. We use hypomorphic and null alleles of Foxc1 to study the effect of meningeal defects on neocortical organization. Methods Embryos with loss of Foxc1 activity were generated using the hypomorphic Foxc1hith allele and the null Foxc1lacZ allele. Immunohistologic analysis was used to assess cerebral basement membrane integrity, marginal zone heterotopia formation, neuronal overmigration, meningeal defects, and changes in basement membrane composition. Dysplasia severity was quantified using two measures. Results Cortical dysplasia resembling cobblestone cortex, with basement membrane breakdown and lamination defects, is seen in Foxc1 mutants. As Foxc1 activity was reduced, abnormalities in basement membrane integrity, heterotopia formation, neuronal overmigration, and meningeal development appeared earlier in gestation and were more severe. Surprisingly, the basement membrane appeared intact at early stages of development in the face of severe deficits in meningeal development. Prominent defects in basement membrane integrity appeared as development proceeded. Molecular analysis of basement membrane laminin subunits demonstrated that loss of the meninges led to changes in basement membrane composition. Interpretation Cortical dysplasia can be caused by cellular defects in the meninges. The meninges are not required for basement membrane establishment but are needed for remodeling as the brain expands. Specific changes in basement membrane composition may contribute to subsequent breakdown. Our study raises the possibility that primary meningeal defects may cortical dysplasia in some cases. PMID:20976766

Sperm membrane functionality in the dog assessed by flow cytometry.

PubMed

Cheuquemán, C; Bravo, P; Treulén, F; Giojalas, Lc; Villegas, J; Sánchez, R; Risopatrón, J

2012-02-01

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa. © 2011 Blackwell Verlag GmbH.

Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity*

PubMed Central

Smith, Ryan C.; King, Jonas G.; Tao, Dingyin; Zeleznik, Oana A.; Brando, Clara; Thallinger, Gerhard G.; Dinglasan, Rhoel R.

2016-01-01

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host. PMID:27624304

Biophysical mechanism of the protective effect of blue honeysuckle (Lonicera caerulea L. var. kamtschatica Sevast.) polyphenols extracts against lipid peroxidation of erythrocyte and lipid membranes.

PubMed

Bonarska-Kujawa, D; Pruchnik, H; Cyboran, S; Żyłka, R; Oszmiański, J; Kleszczyńska, H

2014-07-01

The aim of the present research was to determine the effect of blue honeysuckle fruit and leaf extracts components on the physical properties of erythrocyte and lipid membranes and assess their antioxidant properties. The HPLC analysis showed that the extracts are rich in polyphenol anthocyanins in fruits and flavonoids in leaves. The results indicate that both extracts have antioxidant activity and protect the red blood cell membrane against oxidation induced by UVC irradiation and AAPH. The extracts do not induce hemolysis and slightly increase osmotic resistance of erythrocytes. The research showed that extracts components are incorporated mainly in the external part of the erythrocyte membrane, inducing the formation of echinocytes. The values of generalized polarization and fluorescence anisotropy indicate that the extracts polyphenols alter the packing arrangement of the hydrophilic part of the erythrocyte and lipid membranes, without changing the fluidity of the hydrophobic part. The DSC results also show that the extract components do not change the main phase transition temperature of DPPC membrane. Studies of electric parameters of membranes modified by the extracts showed that they slightly stabilize lipid membranes and do not reduce their specific resistance or capacity. Examination of IR spectra indicates small changes in the degree of hydration in the hydrophilic region of liposomes under the action of the extracts. The location of polyphenolic compounds in the hydrophilic part of the membrane seems to constitute a protective shield of the cell against other substances, the reactive forms of oxygen in particular.

Comparative research of effectiveness of cellulose and fiberglass porous membrane carriers for bio sampling in veterinary and food industry monitoring

NASA Astrophysics Data System (ADS)

Gusev, Alexander; Vasyukova, Inna; Zakharova, Olga; Altabaeva, Yuliya; Saushkin, Nikolai; Samsonova, Jeanne; Kondakov, Sergey; Osipov, Alexander; Snegin, Eduard

2017-11-01

The aim of proposed research is to study the applicability of fiberglass porous membrane materials in a new strip format for dried blood storage in food industry monitoring. A comparative analysis of cellulosic and fiberglass porous membrane materials was carried out to obtain dried samples of serum or blood and the possibility of further species-specific analysis. Blood samples of Sus scrofa were used to study the comparative effectiveness of cellulose and fiberglass porous membrane carriers for long-term biomaterial storage allowing for further DNA detection by real-time polymerase chain reaction (PCR) method. Scanning electron microscopy of various membranes - native and with blood samples - indicate a fundamental difference in the form of dried samples. Membranes based on cellulosic materials sorb the components of the biological fluid on the surface of the fibers of their structure, partially penetrating the cellulose fibers, while in the case of glass fiber membranes the components of the biological fluid dry out as films in the pores of the membrane between the structural filaments. This fundamental difference in the retention mechanisms affects the rate of dissolution of the components of dry samples and contributes to an increase in the efficiency of the desorption process of the sample before subsequent analysis. Detecting of pig DNA in every analyzed sample under the performed Real-time PCR as well as good state of the biomaterial preservation on the glass fiber membranes was clearly demonstrated. Good biomaterials preservation has been revealed on the test cards for 4 days as well as for 1 hour.

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

PubMed Central

Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

PubMed

Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

PubMed

Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

2010-02-01

By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

Short-Term Storage of Rat Sperm in the Presence of Various Extenders

PubMed Central

Varisli, Omer; Agca, Cansu; Agca, Yuksel

2013-01-01

Sperm preservation protocols differ among animal species because of different sperm characteristics among species. Rat sperm have extreme sensitivity to suboptimal conditions in centrifugation, pipetting and chilling due to their longer tail, the shape and size of the sperm head, and membrane composition. The aim of this study was to determine optimal conditions for short-term storage of rat sperm by evaluating their motility and membrane and acrosomal integrity in response to various extender solutions, temperatures, and durations. Motility of rat sperm was highest when stored at 22 °C; motility was 28% and 14% at 72 h in TL-HEPES and PBS extenders, respectively. The motility and membrane integrity of rat sperm fell significantly within 24 h at 4 and 37 °C. Although cold storage did not have a detrimental effect on acrosomal integrity of sperm, room temperature storage reduced acrosomal integrity after 24 h. LEY extender caused the highest loss in acrosomal integrity at 48 and 72 h. In conclusion, storage at 4 or 37 °C reduced the motility and membrane integrity of rat sperm even with short incubation periods. Rat sperm stored in TL-HEPES or PBS remained motile for at least 3 d when held at 22 °C. PMID:24351761

Calculating the permeability coefficients of mixed matrix membranes of polydimethylsiloxane and silicalite crystals to various ethanol-water solutions using molecular simulations.

EPA Science Inventory

The permeability coefficients of mixed matrix membranes of polydimethylsiloxane (PDMS) and silicalite crystal are taken as the sum of the permeability coefficients of membrane components each weighted by their associated mass fraction. The permeability coefficient of a membrane c...

Polymeric and Lipid Membranes—From Spheres to Flat Membranes and vice versa

PubMed Central

Saveleva, Mariia S.; Gorin, Dmitry A.; Skirtach, Andre G.

2017-01-01

Membranes are important components in a number of systems, where separation and control of the flow of molecules is desirable. Controllable membranes represent an even more coveted and desirable entity and their development is considered to be the next step of development. Typically, membranes are considered on flat surfaces, but spherical capsules possess a perfect “infinite” or fully suspended membranes. Similarities and transitions between spherical and flat membranes are discussed, while applications of membranes are also emphasized. PMID:28809796

Fabrication of a silver particle-integrated silicone polymer-covered metal stent against sludge and biofilm formation and stent-induced tissue inflammation

PubMed Central

Lee, Tae Hoon; Jang, Bong Seok; Jung, Min Kyo; Pack, Chan Gi; Choi, Jun-Ho; Park, Do Hyun

2016-01-01

To reduce tissue or tumor ingrowth, covered self-expandable metal stents (SEMSs) have been developed. The effectiveness of covered SEMSs may be attenuated by sludge or stone formation or by stent clogging due to the formation of biofilm on the covering membrane. In this study, we tested the hypothesis that a silicone membrane containing silver particles (Ag-P) would prevent sludge and biofilm formation on the covered SEMS. In vitro, the Ag-P-integrated silicone polymer-covered membrane exhibited sustained antibacterial activity, and there was no definite release of silver ions from the Ag-P-integrated silicone polymer membrane at any time point. Using a porcine stent model, in vivo analysis demonstrated that the Ag-P-integrated silicone polymer-covered SEMS reduced the thickness of the biofilm and the quantity of sludge formed, compared with a conventional silicone-covered SEMS. In vivo, the release of silver ions from an Ag-P-integrated silicone polymer-covered SEMS was not detected in porcine serum. The Ag-P-integrated silicone polymer-covered SEMS also resulted in significantly less stent-related bile duct and subepithelium tissue inflammation than a conventional silicone polymer-covered SEMS. Therefore, the Ag-P-integrated silicone polymer-covered SEMS reduced sludge and biofilm formation and stent-induced pathological changes in tissue. This novel SEMS may prolong the stent patency in clinical application. PMID:27739486

Fabrication of a silver particle-integrated silicone polymer-covered metal stent against sludge and biofilm formation and stent-induced tissue inflammation.

PubMed

Lee, Tae Hoon; Jang, Bong Seok; Jung, Min Kyo; Pack, Chan Gi; Choi, Jun-Ho; Park, Do Hyun

2016-10-14

To reduce tissue or tumor ingrowth, covered self-expandable metal stents (SEMSs) have been developed. The effectiveness of covered SEMSs may be attenuated by sludge or stone formation or by stent clogging due to the formation of biofilm on the covering membrane. In this study, we tested the hypothesis that a silicone membrane containing silver particles (Ag-P) would prevent sludge and biofilm formation on the covered SEMS. In vitro, the Ag-P-integrated silicone polymer-covered membrane exhibited sustained antibacterial activity, and there was no definite release of silver ions from the Ag-P-integrated silicone polymer membrane at any time point. Using a porcine stent model, in vivo analysis demonstrated that the Ag-P-integrated silicone polymer-covered SEMS reduced the thickness of the biofilm and the quantity of sludge formed, compared with a conventional silicone-covered SEMS. In vivo, the release of silver ions from an Ag-P-integrated silicone polymer-covered SEMS was not detected in porcine serum. The Ag-P-integrated silicone polymer-covered SEMS also resulted in significantly less stent-related bile duct and subepithelium tissue inflammation than a conventional silicone polymer-covered SEMS. Therefore, the Ag-P-integrated silicone polymer-covered SEMS reduced sludge and biofilm formation and stent-induced pathological changes in tissue. This novel SEMS may prolong the stent patency in clinical application.

Improved quality of frozen boer goat semen with the addition of sweet orange essential oil on tris yolk and gentamicin extender

NASA Astrophysics Data System (ADS)

Sitepu, S. A.; Zaituni, U.; Jaswandi; Hendri

2018-02-01

This research aimed to determine the extent of frozen semen quality Boer Goat by essential oils of sweet orange peel in tris yolk and gentamicin extender. Research has been conducted at the Laboratory Loka Penelitian Kambing Potong Sei Putih, Deli Serdang, North Sumatra in February 2017. This study used a completely randomized design with 4 treatments and 5 replications. Treatments are 0.25; 0.5; 0.75 and 1% essential oils as additional diluent. The parameters were measured percentage Motility, membrane integrity, acrosome integrity and viability Boer Goat frozen semen. The results showed that the addition of essential oils as diluent semen was significant (P <0.01) in the percentage motility, Viability, membrane integrity and acrosome integrity Boer Goat frozen semen. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group. The best results of all treatments In the study was the addition of essential oil as much as 1%.

Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

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

Y Cao; X Jin; E Levin

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which ismore » occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.« less

Screening antiallergic components from Carthamus tinctorius using rat basophilic leukemia 2H3 cell membrane chromatography combined with high-performance liquid chromatography and tandem mass spectrometry.

PubMed

Han, Shengli; Huang, Jing; Cui, Ronghua; Zhang, Tao

2015-02-01

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high-performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β-hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high-performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E-antigen-mediated degranulation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cholesterol removal from adult skeletal muscle impairs excitation–contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins

PubMed Central

Barrientos, Genaro; Llanos, Paola; Hidalgo, Jorge; Bolaños, Pura; Caputo, Carlo; Riquelme, Alexander; Sánchez, Gina; Quest, Andrew F. G.; Hidalgo, Cecilia

2015-01-01

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation–contraction (E–C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E–C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals. PMID:25914646

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins

PubMed Central

Debelyy, Mykhaylo O.; Waridel, Patrice; Quadroni, Manfredo; Conzelmann, Andreas

2017-01-01

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p). PMID:29073188

Annexins in plasma membrane repair.

PubMed

Boye, Theresa Louise; Nylandsted, Jesper

2016-10-01

Disruption of the plasma membrane poses deadly threat to eukaryotic cells and survival requires a rapid membrane repair system. Recent evidence reveal various plasma membrane repair mechanisms, which are required for cells to cope with membrane lesions including membrane fusion and replacement strategies, remodeling of cortical actin cytoskeleton and vesicle wound patching. Members of the annexin protein family, which are Ca2+-triggered phospholipid-binding proteins emerge as important components of the plasma membrane repair system. Here, we discuss the mechanisms of plasma membrane repair involving annexins spanning from yeast to human cancer cells.

GM1 and GM2 gangliosides: recent developments.

PubMed

Bisel, Blaine; Pavone, Francesco S; Calamai, Martino

2014-03-01

GM1 and GM2 gangliosides are important components of the cell membrane and play an integral role in cell signaling and metabolism. In this conceptual overview, we discuss recent developments in our understanding of the basic biological functions of GM1 and GM2 and their involvement in several diseases. In addition to a well-established spectrum of disorders known as gangliosidoses, such as Tay-Sachs disease, more and more evidence points at an involvement of GM1 in Alzheimer's and Parkinson's diseases. New emerging methodologies spanning from single-molecule imaging in vivo to simulations in silico have complemented standard studies based on ganglioside extraction.

Cholesterol: a novel regulatory role in myelin formation.

PubMed

Saher, Gesine; Quintes, Susanne; Nave, Klaus-Armin

2011-02-01

Myelin consists of tightly compacted membranes that form an insulating sheath around axons. The function of myelin for rapid saltatory nerve conduction is dependent on its unique composition, highly enriched in glycosphingolipids and cholesterol. Cholesterol emerged as the only integral myelin component that is essential and rate limiting for the development of CNS and PNS myelin. Experiments with conditional mouse mutants that lack cholesterol biosynthesis in oligodendrocytes revealed that only minimal changes of the CNS myelin lipid composition are tolerated. In Schwann cells of the PNS, protein trafficking and myelin compaction depend on cholesterol. In this review, the authors summarize the role of cholesterol in myelin biogenesis and myelin disease.

Handheld colorimeter for determination of heavy metal concentrations

NASA Astrophysics Data System (ADS)

López Ruiz, N.; Ariza, M.; Martínez Olmos, A.; Vukovic, J.; Palma, A. J.; Capitan-Vallvey, L. F.

2011-08-01

A portable instrument that measures heavy metal concentration from a colorimetric sensor array is presented. The use of eight sensing membranes, placed on a plastic support, allows to obtain the hue component of the HSV colour space of each one in order to determinate the concentration of metals present in a solution. The developed microcontroller-based system captures, in an ambient light environment, an image of the sensor array using an integrated micro-camera and shows the picture in a touch micro-LCD screen which acts as user interface. After image-processing of the regions of interest selected by the user, colour and concentration information are displayed on the screen.

A study of oxidative stress induced by non-thermal plasma-activated water for bacterial damage

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

Zhang, Qian; Ma, Ruonan; Tian, Ying

2013-05-20

Ar/O{sub 2} (2%) cold plasma microjet was used to create plasma-activated water (PAW). The disinfection efficacy of PAW against Staphylococcus aureus showed that PAW can effectively disinfect bacteria. Optical emission spectra and oxidation reduction potential results demonstrated the inactivation is attributed to oxidative stress induced by reactive oxygen species in PAW. Moreover, the results of X-ray photoelectron spectroscopy, atomic absorption spectrometry, and transmission electron microscopy suggested that the chemical state of cell surface, the integrity of cell membrane, as well as the cell internal components and structure were damaged by the oxidative stress.

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

PubMed

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

The vascular basement membrane in the healthy and pathological brain.

PubMed

Thomsen, Maj S; Routhe, Lisa J; Moos, Torben

2017-10-01

The vascular basement membrane contributes to the integrity of the blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs). The BCECs receive support from pericytes embedded in the vascular basement membrane and from astrocyte endfeet. The vascular basement membrane forms a three-dimensional protein network predominantly composed of laminin, collagen IV, nidogen, and heparan sulfate proteoglycans that mutually support interactions between BCECs, pericytes, and astrocytes. Major changes in the molecular composition of the vascular basement membrane are observed in acute and chronic neuropathological settings. In the present review, we cover the significance of the vascular basement membrane in the healthy and pathological brain. In stroke, loss of BBB integrity is accompanied by upregulation of proteolytic enzymes and degradation of vascular basement membrane proteins. There is yet no causal relationship between expression or activity of matrix proteases and the degradation of vascular matrix proteins in vivo. In Alzheimer's disease, changes in the vascular basement membrane include accumulation of Aβ, composite changes, and thickening. The physical properties of the vascular basement membrane carry the potential of obstructing drug delivery to the brain, e.g. thickening of the basement membrane can affect drug delivery to the brain, especially the delivery of nanoparticles.

Tannin-rich fraction from pomegranate rind damages membrane of Listeria monocytogenes.

PubMed

Li, Guanghui; Xu, Yunfeng; Wang, Xin; Zhang, Baigang; Shi, Chao; Zhang, Weisong; Xia, Xiaodong

2014-04-01

Pomegranate rind has been reported to inhibit several foodborne pathogens, and its antimicrobial activity has been attributed mainly to its tannin fraction. This study aimed to investigate the antimicrobial activity of the tannin-rich fraction from pomegranate rind (TFPR) against Listeria monocytogenes and its mechanism of action. The tannin-related components of TFPR were analyzed by high-performance liquid chromatography and liquid chromatography-mass spectrometry, and the minimum inhibitory concentration (MIC) of TFPR was determined using the agar dilution method. Extracellular potassium concentration, the release of cell constituents, intra- and extracellular ATP concentrations, membrane potential, and intracellular pH (pHin) were measured to elucidate a possible antibacterial mechanism. Punicalagin (64.2%, g/g) and ellagic acid (3.1%, g/g) were detected in TFPR, and the MICs of TFPR were determined to be 1.25-5.0 mg/mL for different L. monocytogenes strains. Treatment with TFPR induced a decrease of the intracellular ATP concentration, an increase of the extracellular concentrations of potassium and ATP, and the release of cell constituents. A reduction of pHin and cell membrane hyperpolarization were observed after treatment. Electron microscopic observations showed that the cell membrane structures of L. monocytogenes were apparently impaired by TFPR. It is concluded that TFPR could destroy the integrity of the cell membrane of L. monocytogenes, leading to a loss of cell homeostasis. These findings indicate that TFPR has the potential to be used as a food preservative in order to control L. monocytogenes contamination in food and reduce the risk of listeriosis.

Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques.

PubMed

Velikova, Violeta; Várkonyi, Zsuzsanna; Szabó, Milán; Maslenkova, Liliana; Nogues, Isabel; Kovács, László; Peeva, Violeta; Busheva, Mira; Garab, Gyozo; Sharkey, Thomas D; Loreto, Francesco

2011-10-01

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA₅₁₅) was evaluated following single-turnover saturating flashes. The decay of ΔA₅₁₅ was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S₂Q(B)⁻ charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S₂Q(B)⁻ redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses.

Increased Thermostability of Thylakoid Membranes in Isoprene-Emitting Leaves Probed with Three Biophysical Techniques1[W][OA

PubMed Central

Velikova, Violeta; Várkonyi, Zsuzsanna; Szabó, Milán; Maslenkova, Liliana; Nogues, Isabel; Kovács, László; Peeva, Violeta; Busheva, Mira; Garab, Győző; Sharkey, Thomas D.; Loreto, Francesco

2011-01-01

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA515) was evaluated following single-turnover saturating flashes. The decay of ΔA515 was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S2QB− charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S2QB− redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses. PMID:21807886

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.

Flow cytometry immunodetection and membrane integrity assessment of Escherichia coli O157:H7 in ready-to-eat pasta salad during refrigerated storage.

PubMed

Subires, Alicia; Yuste, Josep; Capellas, Marta

2014-01-03

Over the past years, products of non-animal origin have been increasingly linked to foodborne diseases caused by the enterohemorrhagic pathogen Escherichia coli O157:H7. Contaminated fresh produce and derived ready-to-eat meals are of major concern, since no further or only minimal processing is applied. In this study, flow cytometry was evaluated as a rapid technique to detect E. coli O157:H7 by immunofluorescence, using polyclonal antibodies conjugated to R-phycoerythrin, in refrigerated ready-to-eat pasta salad containing acetic acid and benzoic acid. Signal filtering strategies were applied during sample analysis to reduce the limit of detection of the technique to 5 log CFU/g. Simultaneously with pathogen detection, physiological state was assessed by staining with the membrane integrity indicators propidium iodide and SYBR Green I. Fine tuning of dye concentrations and ratios allowed discrimination of not only cells with intact or damaged membranes, but also of cells with partially damaged membranes, which were considered injured cells. Then, changes in membrane integrity of inoculated E. coli O157:H7 cells were monitored throughout 14-day refrigerated storage. Most cells were injured at the beginning of refrigeration, but showed an intact membrane at the end. This suggests that injured E. coli O157:H7 cells underwent a membrane repair during exposure to refrigeration and acid stresses, and survived in ready-to-eat pasta salad. This highlights the importance of the implementation of control measures to limit the presence of this pathogen in non-animal origin food products. Additionally, the proposed immunodetection and membrane integrity three-color assay in food is a good tool to monitor the effect of a number of food-related treatments on E. coli O157:H7 cell membrane. © 2013.

Foreign body giant cells selectively covering haptics of intraocular lens implants: indicators of poor toleration?

PubMed

Wolter, J R

1983-10-01

A Sputnik lens implant removed after five years because of bullous keratopathy exhibits a dense covering of its Supramid anterior staves with large foreign body giant cells, while its Prolene loops and Polymethylmethacrylate optics have attracted only few of these cell units. The glass-membrane-like component of the reactive membrane also shows significant differences on the different parts of this implant. The use of observation of the components of reactive membranes on lens implants as indicators of toleration in the eye is suggested.

Shaping the Flavivirus Replication Complex: It's Curvaceous!

PubMed

Aktepe, Turgut E; Mackenzie, Jason M

2018-06-22

Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus-induced organelles characteristic of infection. This article is protected by copyright. All rights reserved.

The new approaches to preservation of graft cell integrity in preservation for transplantation.

PubMed

Gewartowska, Magdalena; Olszewski, Waldemar L

2005-01-01

Restoration of cell plasma membrane integrity after injury is essential for the survival of animal cells. In case of graft preservation or during chemotherapy in cancer, cell membrane integrity and the process of its repair are disrupted. Cytoprotective substances are important in such cases, as well as in other diseases, for example in myocardial infarction, acute insults and in chronic neurodegenerative diseases. Hyperosmolarity is a condition in which cell membrane stability may be damaged in vivo but preserved in the in vitro conditions. Hypertonicity causes water leaving from cells by osmosis, decreasing cell volume and increasing of intracellular ionic strength. High intracellular ionic strength perturbs cellular function by decreasing the rates of biochemical reaction. We review the new experimentally studied cytoprotective substances and their application in cell membrane protection. Moreover, we present our data on the effects of hyperosmolarity and its protective effect on cell internal structure.

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

PubMed

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Neutrons Provide the First Nanoscale Look at a Living Cell Membrane

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

None

Neutron scattering is a valuable technique for studying cell membranes, but signals from the cell’s other components such as proteins, RNA, DNA and carbohydrates can get in the way. An ORNL team made these other components practically invisible to neutrons by combining specific levels of heavy hydrogen (deuterium) with normal hydrogen within the cell.

Real Time Measures of Prestin Charge and Fluorescence during Plasma Membrane Trafficking Reveal Sub-Tetrameric Activity

PubMed Central

Bian, Shumin; Navaratnam, Dhasakumar; Santos-Sacchi, Joseph

2013-01-01

Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional prestin is not an obligate tetramer. PMID:23762468

Identification of minor inner-membrane components of the Shigella type III secretion system 'needle complex'.

PubMed

Zenk, Sebastian F; Stabat, David; Hodgkinson, Julie L; Veenendaal, Andreas K J; Johnson, Steven; Blocker, Ariel J

2007-08-01

Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the 'needle complex' (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a 'pilotin'-type protein for the outer-membrane 'secretin' ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.

Measurements of three-dimensional shape and sound-induced motion of the chinchilla tympanic membrane

PubMed Central

Rosowski, John J; Dobrev, Ivo; Khaleghi, Morteza; Lu, Weina; Cheng, Jeffrey Tao; Harrington, Ellery; Furlong, Cosme

2013-01-01

Opto-electronic computer holographic measurements were made of the tympanic membrane (TM) in cadaveric chinchillas. Measurements with two laser wavelengths were used to compute the 3D-shape of the TM. Single laser wavelength measurements locked to eight distinct phases of a tonal stimulus were used to determine the magnitude and the relative phase of the surface displacements. These measurements were made at over 250,000 points on the TM surface. The measured motions contained spatial phase variations consistent with relatively low-order (large spatial frequency) modal motions and smaller magnitude higher-order (smaller spatial frequency) motions that appear to travel, but may also be explained by losses within the membrane. The measurement of shape and thin shell theory allowed us to separate the measured motions into those components orthogonal to the plane of the tympanic ring, and those components within the plane of the tympanic ring based on the 3D-shape. The predicted in-plane motion components are generally smaller than the out-of-plane perpendicular component of motion. Since the derivation of in-plane and out-of plane depended primarily on the membrane shape, the relative sizes of the predicted motion components did not vary with frequency. PMID:23247058

Vanadium(V) Reduction by Shewanella oneidensis MR-1 Requires Menaquinone and Cytochromes from the Cytoplasmic and Outer Membranes

PubMed Central

Myers, Judith M.; Antholine, William E.; Myers, Charles R.

2004-01-01

The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species. PMID:15006760

Dual phase high-temperature membranes for CO2 separation - performance assessment in post- and pre-combustion processes.

PubMed

Anantharaman, Rahul; Peters, Thijs; Xing, Wen; Fontaine, Marie-Laure; Bredesen, Rune

2016-10-20

Dual phase membranes are highly CO 2 -selective membranes with an operating temperature above 400 °C. The focus of this work is to quantify the potential of dual phase membranes in pre- and post-combustion CO 2 capture processes. The process evaluations show that the dual phase membranes integrated with an NGCC power plant for CO 2 capture are not competitive with the MEA process for post-combustion capture. However, dual phase membrane concepts outperform the reference Selexol technology for pre-combustion CO 2 capture in an IGCC process. The two processes evaluated in this work, post-combustion NGCC and pre-combustion IGCC, represent extremes in CO 2 partial pressure fed to the separation unit. Based on the evaluations it is expected that dual phase membranes could be competitive for post-combustion capture from a pulverized coal fired power plant (PCC) and pre-combustion capture from an Integrated Reforming Cycle (IRCC).

Medical telesensors

NASA Astrophysics Data System (ADS)

Ferrell, Trinidad L.; Crilly, P. B.; Smith, S. F.; Wintenberg, Alan L.; Britton, Charles L., Jr.; Morrison, Gilbert W.; Ericson, M. N.; Hedden, D.; Bouldin, Donald W.; Passian, A.; Downey, Todd R.; Wig, A. G.; Meriaudeau, Fabrice

1998-05-01

Medical telesensors are self-contained integrated circuits for measuring and transmitting vital signs over a distance of approximately 1-2 meters. The circuits are unhoused and contain a sensor, signal processing and modulation electronics, a spread-spectrum transmitter, an antenna and a thin-film battery. We report on a body-temperature telesensor, which is sufficiently small to be placed on a tympanic membrane in a child's ear. We also report on a pulse-oximeter telesensor and a micropack receiver/long- range transmitter unit, which receives form a telesensor array and analyzes and re-transmits the vital signs over a longer range. Signal analytics are presented for the pulse oximeter, which is currently in the form of a finger ring. A multichip module is presented as the basic signal-analysis component. The module contains a microprocessor, a field=programmable gate array, memory elements and other components necessary for determining trauma and reporting signals.

Development of a lightweight fuel cell vehicle

NASA Astrophysics Data System (ADS)

Hwang, J. J.; Wang, D. Y.; Shih, N. C.

This paper described the development of a fuel cell system and its integration into the lightweight vehicle known as the Mingdao hydrogen vehicle (MHV). The fuel cell system consists of a 5-kW proton exchange membrane fuel cell (PEMFC), a microcontroller and other supported components like a compressed hydrogen cylinder, blower, solenoid valve, pressure regulator, water pump, heat exchanger and sensors. The fuel cell not only propels the vehicle but also powers the supporting components. The MHV performs satisfactorily over a hundred-kilometer drive thus validating the concept of a fuel cell powered zero-emission vehicle. Measurements further show that the fuel cell system has an efficiency of over 30% at the power consumption for vehicle cruise, which is higher than that of a typical internal combustion engine. Tests to improve performance such as speed enhancement, acceleration and fuel efficiency will be conducted in the future work. Such tests will consist of hybridizing with a battery pack.

Ethanol and thermotolerance in the bioconversion of xylose by yeasts

Treesearch

Thomas W. Jeffries; Yong-Su Jin

2000-01-01

The mechanisms underlying ethanol and heat tolerance are complex. Many different genes are involved, and the exact basis is not fully understood. The integrity of cytoplasmic and mitochondrial membranes is critical to maintain proton gradients for metabolic energy and nutrient uptake. Heat and ethanol stress adversely affect membrane integrity. These factors are...

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

PubMed

Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D

2017-12-01

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

PubMed

Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2017-10-01

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Fouling and long-term durability of an integrated forward osmosis and membrane distillation system.

PubMed

Husnain, T; Mi, B; Riffat, R

2015-01-01

An integrated forward osmosis (FO) and membrane distillation (MD) system has great potential for sustainable wastewater reuse. However, the fouling and long-term durability of the system remains largely unknown. This study investigates the fouling behaviour and efficiency of cleaning procedures of FO and MD membranes used for treating domestic wastewater. Results showed that a significant decline in flux of both FO and MD membranes were observed during treatment of wastewater with organic foulants. However, shear force generated by the increased cross-flow physically removed the loosely attached foulants from the FO membrane surface and resulted in 86-88% recovery of flux by cleaning with tap water. For the MD membrane, almost no flux recovery was achieved due to adsorption of organic foulants on the hydrophobic membrane surface, thus indicating significant irreversible fouling/wetting, which may not be effectively cleaned even with chemical reagents. Long-term (10 d) tests showed consistent performance of the FO membrane by rejecting the contaminants. However, organic foulants reduced the hydrophobicity of the MD membrane, caused wetting problems and allowed contaminants to pass through. The results demonstrate that combination of the FO and MD processes can effectively reduce irreversible membrane fouling and solve the wetting problem of the MD membrane.

Modeling a Membrane: Using Engineering Design to Simulate Cell Transport Processes

ERIC Educational Resources Information Center

Mason, Kevin; Evans, Brian

2017-01-01

The "plasma membrane," which controls what comes in and goes out of a cell, is integral to maintaining homeostasis. Cell transport of small molecules across the cell membrane happens in several different ways. Some small, nonpolar molecules cross the plasma membrane along the concentration gradient directly through the "phospholipid…

Protein quality control at the inner nuclear membrane

PubMed Central

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

2015-01-01

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

A micromachined membrane-based active probe for biomolecular mechanics measurement

NASA Astrophysics Data System (ADS)

Torun, H.; Sutanto, J.; Sarangapani, K. K.; Joseph, P.; Degertekin, F. L.; Zhu, C.

2007-04-01

A novel micromachined, membrane-based probe has been developed and fabricated as assays to enable parallel measurements. Each probe in the array can be individually actuated, and the membrane displacement can be measured with high resolution using an integrated diffraction-based optical interferometer. To illustrate its application in single-molecule mechanics experiments, this membrane probe was used to measure unbinding forces between L-selectin reconstituted in a polymer-cushioned lipid bilayer on the probe membrane and an antibody adsorbed on an atomic force microscope cantilever. Piconewton range forces between single pairs of interacting molecules were measured from the cantilever bending while using the membrane probe as an actuator. The integrated diffraction-based optical interferometer of the probe was demonstrated to have <10 fm Hz-1/2 noise floor for frequencies as low as 3 Hz with a differential readout scheme. With soft probe membranes, this low noise level would be suitable for direct force measurements without the need for a cantilever. Furthermore, the probe membranes were shown to have 0.5 µm actuation range with a flat response up to 100 kHz, enabling measurements at fast speeds.

Transforming growth factor β family members in regulation of vascular function: in the light of vascular conditional knockouts.

PubMed

Jakobsson, Lars; van Meeteren, Laurens A

2013-05-15

Blood vessels are composed of endothelial cells, mural cells (smooth muscle cells and pericytes) and their shared basement membrane. During embryonic development a multitude of signaling components orchestrate the formation of new vessels. The process is highly dependent on correct dosage, spacing and timing of these signaling molecules. As vessels mature some cascades remain active, albeit at very low levels, and may be reactivated upon demand. Members of the Transforming growth factor β (TGF-β) protein family are strongly engaged in developmental angiogenesis but are also regulators of vascular integrity in the adult. In humans various genetic alterations within this protein family cause vascular disorders, involving disintegration of vascular integrity. Here we summarize and discuss recent data gathered from conditional and endothelial cell specific genetic loss-of-function of members of the TGF-β family in the mouse. Copyright © 2013 Elsevier Inc. All rights reserved.

Smart polymer brush nanostructures guide the self-assembly of pore-spanning lipid bilayers with integrated membrane proteins

NASA Astrophysics Data System (ADS)

Wilhelmina de Groot, G.; Demarche, Sophie; Santonicola, M. Gabriella; Tiefenauer, Louis; Vancso, G. Julius

2014-01-01

Nanopores in arrays on silicon chips are functionalized with pH-responsive poly(methacrylic acid) (PMAA) brushes and used as supports for pore-spanning lipid bilayers with integrated membrane proteins. Robust platforms are created by the covalent grafting of polymer brushes using surface-initiated atom transfer radical polymerization (ATRP), resulting in sensor chips that can be successfully reused over several assays. His-tagged proteins are selectively and reversibly bound to the nitrilotriacetic acid (NTA) functionalization of the PMAA brush, and consequently lipid bilayer membranes are formed. The enhanced membrane resistance as determined by electrochemical impedance spectroscopy and free diffusion of dyed lipids observed as fluorescence recovery after photobleaching confirmed the presence of lipid bilayers. Immobilization of the His-tagged membrane proteins on the NTA-modified PMAA brush near the pore edges is characterized by fluorescence microscopy. This system allows us to adjust the protein density in free-standing bilayers, which are stabilized by the polymer brush underneath. The potential application of the integrated platform for ion channel protein assays is demonstrated.

The membrane-topogenic vectorial behaviour of Nrf1 controls its post-translational modification and transactivation activity.

PubMed

Zhang, Yiguo; Hayes, John D

2013-01-01

The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression.

LDRD final report on imaging self-organization of proteins in membranes by photocatalytic nano-tagging.

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

Zavadil, Kevin Robert; Shelnutt, John Allen; Sasaki, Darryl Yoshio

We have developed a new nanotagging technology for detecting and imaging the self-organization of proteins and other components of membranes at nanometer resolution for the purpose of investigating cell signaling and other membrane-mediated biological processes. We used protein-, lipid-, or drug-bound porphyrin photocatalysts to grow in-situ nanometer-sized metal particles, which reveal the location of the porphyrin-labeled molecules by electron microscopy. We initially used photocatalytic nanotagging to image assembled multi-component proteins and to monitor the distribution of lipids and porphyrin labels in liposomes. For example, by exchanging the heme molecules in hemoproteins with a photocatalytic tin porphyrin, a nanoparticle was grownmore » at each heme site of the protein. The result obtained from electron microscopy for a tagged multi-subunit protein such as hemoglobin is a symmetric constellation of a specific number of nanoparticle tags, four in the case of the hemoglobin tetramer. Methods for covalently linking photocatalytic porphyrin labels to lipids and proteins were also developed to detect and image the self-organization of lipids, protein-protein supercomplexes, and membrane-protein complexes. Procedures for making photocatalytic porphyrin-drug, porphyrin-lipid, and porphyrin-protein hybrids for non-porphyrin-binding proteins and membrane components were pursued and the first porphyrin-labeled lipids was investigated in liposomal membrane models. Our photocatalytic nanotagging technique may ultimately allow membrane self-organization and cell signaling processes to be imaged in living cells. Fluorescence and plasmonic spectra of the tagged proteins might also provide additional information about protein association and membrane organization. In addition, a porphyrin-aspirin or other NSAID hybrid may be used to grow metal nanotags for the pharmacologically important COX enzymes in membranes so that the distribution of the protein can be imaged at the nanometer scale.« less

Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones

PubMed Central

Linares-Clemente, Pedro; Rozas, José L; Mircheski, Josif; García-Junco-Clemente, Pablo; Martínez-López, José A; Nieto-González, José L; Vázquez, M Eugenio; Pintado, C Oscar; Fernández-Chacón, Rafael

2015-01-01

Key points Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin–phospholipid interaction. Abstract Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB. PMID:25981717

Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones.

PubMed

Linares-Clemente, Pedro; Rozas, José L; Mircheski, Josif; García-Junco-Clemente, Pablo; Martínez-López, José A; Nieto-González, José L; Vázquez, M Eugenio; Pintado, C Oscar; Fernández-Chacón, Rafael

2015-07-01

Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin-phospholipid interaction. Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP*

PubMed Central

Juillan-Binard, Céline; Picciocchi, Antoine; Andrieu, Jean-Pierre; Petit-Hartlein, Isabelle; Caux-Thang, Christelle; Vivès, Corinne; Nivière, Vincent

2017-01-01

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli. We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins. PMID:28028176

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

PubMed Central

Alonso, Hernan

2012-01-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C12E8]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-d-maltopyranoside (DM), n-dodecyl-β-d-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism. PMID:22941083

Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

PubMed

Gadelha, Catarina; Zhang, Wenzhu; Chamberlain, James W; Chait, Brian T; Wickstead, Bill; Field, Mark C

2015-07-01

Surface membrane organization and composition is key to cellular function, and membrane proteins serve many essential roles in endocytosis, secretion, and cell recognition. The surface of parasitic organisms, however, is a double-edged sword; this is the primary interface between parasites and their hosts, and those crucial cellular processes must be carried out while avoiding elimination by the host immune defenses. For extracellular African trypanosomes, the surface is partitioned such that all endo- and exocytosis is directed through a specific membrane region, the flagellar pocket, in which it is thought the majority of invariant surface proteins reside. However, very few of these proteins have been identified, severely limiting functional studies, and hampering the development of potential treatments. Here we used an integrated biochemical, proteomic and bioinformatic strategy to identify surface components of the human parasite Trypanosoma brucei. This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival. Molecules with receptor-like properties are almost exclusively parasite-specific, whereas transporter-like proteins are conserved in model organisms. Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket. Moreover, the largest systematic analysis of trypanosome surface molecules to date provides evidence that the cell surface is compartmentalized into three distinct domains with free diffusion of molecules in each, but selective, asymmetric traffic between. This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

PubMed

Spicer, S Timothy; Tran, Giang T; Killingsworth, Murray C; Carter, Nicole; Power, David A; Paizis, Kathy; Boyd, Rochelle; Hodgkinson, Suzanne J; Hall, Bruce M

2007-07-01

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

PubMed

Maerker, Tina; van Wijk, Erwin; Overlack, Nora; Kersten, Ferry F J; McGee, Joann; Goldmann, Tobias; Sehn, Elisabeth; Roepman, Ronald; Walsh, Edward J; Kremer, Hannie; Wolfrum, Uwe

2008-01-01

The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.

Fouling analysis of membrane bioreactor treating antibiotic production wastewater at different hydraulic retention times.

PubMed

Yu, Dawei; Chen, Yutao; Wei, Yuansong; Wang, Jianxing; Wang, Yawei; Li, Kun

2017-04-01

Membrane fouling, including foulants and factors, was investigated during hydraulic retention time (HRT) optimization of a membrane bioreactor (MBR) that treated wastewater from the production of antibiotics. The results showed that HRT played an important role in membrane fouling. Trans-membrane pressure (TMP), membrane flux, and resistance were stable at -6 kPa, 76 L m -2  h -1  bar -1 , and 4.5 × 10 12  m -1 when HRT was at 60, 48, and 36 h, respectively. Using Fourier transform infrared spectroscopy, foulants were identified as carbohydrates and proteins, which correlated with effluent organic matter and effluent chemical oxygen demand (COD) compounds. Therefore, membrane fouling trends would benefit from low supernatant COD (378 mg L -1 ) and a low membrane removal rate (26 %) at a HRT of 36 h. Serious membrane fouling at 72 and 24 h was related to soluble microbial products and extracellular polymeric substances in mixed liquor, respectively. Based on the TMP decrease and flux recovery after physical and chemical cleaning, irremovable fouling aggravation was related to extracellular polymeric substances' increase and soluble microbial products' decrease. According to changes in the specific oxygen uptake rate (SOUR) and mixed liquor suspended solids (MLSSs) during HRT optimization in this study, antibiotic production wastewater largely inhibited MLSS growth, which only increased from 4.5 to 5.0 g L -1 when HRT was decreased from 72 to 24 h, but did not limit sludge activity. The results of a principal component analysis highlighted both proteins and carbohydrates in extracellular polymeric substances as the primary foulants. Membrane fouling associated with the first principal component was positively related to extracellular polymeric substances and negatively related to soluble microbial products. Principal component 2 was primarily related to proteins in the influent. Additional membrane fouling factors included biomass characteristics, operational conditions, and feed characteristics.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

Correlated alterations in prostate basal cell layer and basement membrane

PubMed Central

Liu, Aijun; Wei, Lixin; Gardner, William A.; Deng, Chu-Xia; Man, Yan-Gao

2009-01-01

Our recent studies revealed that focal basal cell layer disruption (FBCLD) induced auto-immunoreactions represented a contributing factor for human prostate tumor progression and invasion. As the basement membrane surrounds and attaches to the basal cell layer, our current study assessed whether FBCLD would impact the physical integrity of the associated basement membrane. Paraffin sections from 25-human prostate tumors were subjected to double immunohistochemistry to simultaneously elucidate the basal cell layer and the basement membrane with corresponding biomarkers. The physical integrity of the basement membrane overlying FBCLD was examined to determine the extent of correlated alterations. Of a total of 89 FBCLD encountered, 76 (85 %) showed correlated alterations in the overlying basement membrane, which included distinct focal disruptions or fragmentations. In the remaining 13 (15%) FBCLD, the overlying basement membrane showed significant attenuation or reduction of the immunostaining intensity. The basement membrane in all or nearly all ducts or acini with p63 positive basal cells was substantially thicker and more uniform than that in ducts or acini without p63 positive basal cells, and also, a vast majority of the focal disruptions occurred near basal cells that lack p63 expression. These findings suggest that focal disruptions in the basal cell layer and alterations in the basement membrane are correlated events and that the physical and functional status of the basal cells could significantly impact the physical integrity of the overlying basement membrane. As the degradation of both the basal cell layer and the basement membrane is a pre-requisite for prostate tumor invasion or progression, ducts or acini with focally disrupted basal cell layer and basement membrane are likely at greater risk to develop invasive lesions. Thus, further elucidation of the specific molecules and mechanism associated with these events may lead to the development of a more effective alternative for repeat biopsy to monitor tumor progression and invasion. PMID:19343113

Process for recycling components of a PEM fuel cell membrane electrode assembly

DOEpatents

Shore, Lawrence [Edison, NJ

2012-02-28

The membrane electrode assembly (MEA) of a PEM fuel cell can be recycled by contacting the MEA with a lower alkyl alcohol solvent which separates the membrane from the anode and cathode layers of the assembly. The resulting solution containing both the polymer membrane and supported noble metal catalysts can be heated under mild conditions to disperse the polymer membrane as particles and the supported noble metal catalysts and polymer membrane particles separated by known filtration means.

Identification of Key Interactions in the Initial Self-Assembly of Amylin in a Membrane Environment.

PubMed

Christensen, Mikkel; Skeby, Katrine K; Schiøtt, Birgit

2017-09-12

Islet amyloid polypeptide, also known as amylin, forms aggregates that reduce the amount of insulin-producing cells in patients with type II diabetes mellitus. Much remains unknown about the process of aggregation and cytotoxicity, but it is known that certain cell membrane components can alter the rate of aggregation. Using atomistic molecular dynamics simulations combined with the highly mobile membrane mimetic model incorporating enhanced sampling of lipid diffusion, we investigate interaction of amylin peptides with the membrane components as well as the self-assembly of amylin. Consistent with experimental evidence, we find that an initial membrane-bound α-helical state folds into stable β-sheet structures upon self-assembly. Our results suggest the following mechanism for the initial phase of amylin self-assembly. The peptides move around on the membrane with the positively charged N-terminus interacting with the negatively charged lipid headgroups. When the peptides start to interact, they partly unfold and break some of the contacts with the membrane. The initial interactions between the peptides are dominated by aromatic and hydrophobic interactions. Oligomers are formed showing both intra- and interpeptide β-sheets, initially with interactions mainly in the C-terminal domain of the peptides. Decreasing the pH to 5.5 is known to inhibit amyloid formation. At low pH, His18 is protonated, adding a fourth positive charge at the peptide. With His18 protonated, no oligomerization is observed in the simulations. The additional charge gives a strong midpoint anchoring of the peptides to negatively charged membrane components, and the peptides experience additional interpeptide repulsion, thereby preventing interactions.

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Strassburg, Sandra; Hodson, Nigel W; Hill, Patrick I; Richardson, Stephen M; Hoyland, Judith A

2012-01-01

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

Preparation of pH-sensitive anionic liposomes designed for drug delivery system (DDS) application.

PubMed

Aoki, Asami; Akaboshi, Hikaru; Ogura, Taku; Aikawa, Tatsuo; Kondo, Takeshi; Tobori, Norio; Yuasa, Makoto

2015-01-01

We prepared pH-sensitive anionic liposomes composed solely of anionic bilayer membrane components that were designed to promote efficient release of entrapped agents in response to acidic pH. The pH-sensitive anionic liposomes showed high dispersion stability at neutral pH, but the fluidity of the bilayer membrane was enhanced in an acidic environment. These liposomes were rather simple and were composed of dimyristoylphosphatidylcholine (DMPC), an anionic bilayer membrane component, and polyoxyethylene sorbitan monostearate (Tween 80). In particular, the present pH-sensitive anionic liposomes showed higher temporal stability than those of conventional DMPC/DPPC liposomes. We found that pHsensitive properties strongly depended on the molecular structure, pKa value, and amount of an incorporated anionic bilayer membrane component, such as sodium oleate (SO), dimyristoylphosphatidylserine (DMPS), or sodium β-sitosterol sulfate (SS). These results provide an opportunity to manipulate liposomal stability in a pH-dependent manner, which could lead to the formulation of a high performance drug delivery system (DDS).

Nanoengineered membrane electrode assembly interface

DOEpatents

Song, Yujiang; Shelnutt, John A

2013-08-06

A membrane electrode structure suitable for use in a membrane electrode assembly (MEA) that comprises membrane-affixed metal nanoparticles whose formation is controlled by a photochemical process that controls deposition of the metal nanoparticles using a photocatalyst integrated with a polymer electrolyte membrane, such as an ionomer membrane. Impregnation of the polymer membrane with the photocatalyst prior to metal deposition greatly reduces the required amount of metal precursor in the deposition reaction solution by restricting metal reduction substantially to the formation of metal nanoparticles affixed on or near the surface of the polymer membrane with minimal formation of metallic particles not directly associated with the membrane.

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.

Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus

PubMed Central

2010-01-01

Background We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates. Methods We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity. Results Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates. Conclusions These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells. PMID:20846374

Autoimmune Subepidermal Bullous Diseases of the Skin and Mucosae: Clinical Features, Diagnosis, and Management.

PubMed

Amber, Kyle T; Murrell, Dedee F; Schmidt, Enno; Joly, Pascal; Borradori, Luca

2018-02-01

Autoimmune subepidermal blistering diseases of the skin and mucosae constitute a large group of sometimes devastating diseases, encompassing bullous pemphigoid, gestational pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, and anti-p200 pemphigoid. Their clinical presentation is polymorphic. These autoimmune blistering diseases are associated with autoantibodies that target distinct components of the basement membrane zone of stratified epithelia. These autoantigens represent structural proteins important for maintenance of dermo-epidermal integrity. Bullous pemphigoid (BP) is the most common subepidermal autoimmune blistering disease of the skin and mucosae. Although the disease typically presents with a generalized blistering eruption associated with itch, atypical variants with either localized bullous lesions or "non-bullous" presentations are observed in approximately 20% of patients. A peculiar form of BP typically associated with pregnancy is pemphigoid gestationis. In anti-p200 pemphigoid, patients present with tense blisters on erythematosus or normal skin resembling BP, with a predilection for acral surfaces. These patients have antibodies targeting the 200-kDa basement membrane protein. Epidermolysis bullosa is a rare autoimmune blistering disease associated with autoantibodies against type VII collagen that can have several phenotypes including a classical form mimicking dystrophic epidermolysis bullosa, an inflammatory presentation mimicking BP, or mucous membrane pemphigoid-like lesions. Mucous membrane pemphigoid (MMP) is the term agreed upon by international consensus for an autoimmune blistering disorder, which affects one or more mucous membrane and may involve the skin. The condition involves a number of different autoantigens in the basement membrane zone. It may result in severe complications from scarring, such as blindness and strictures. Diagnosis of these diseases relies on direct immunofluorescence microscopy studies and immunoserological assays. Management of affected patients is often challenging. We will here review the clinical and immunopathological features as well as the pathophysiology of this group of organ-specific autoimmune diseases. Finally, we will discuss the diagnostic approach and the principles of management in clinical practice.

Poisoning of mixed matrix membranes by fermentation components in pervaporation of ethanol

USDA-ARS?s Scientific Manuscript database

Pervaporation is an alternative to distillation for recovering ethanol produced by fermentation of grains and biomass. Ethanol-selective mixed matrix membranes of the hydrophobic zeolite ZSM-5 in polydimethylsiloxane (PDMS) have superior performance compared to pure PDMS membranes in pervaporation o...

Uncovering homo-and hetero-interactions on the cell membrane using single particle tracking approaches

NASA Astrophysics Data System (ADS)

Torreno-Pina, Juan A.; Manzo, Carlo; Garcia-Parajo, Maria F.

2016-03-01

The plasma membrane of eukaryotic cells is responsible for a myriad of functions that regulate cell physiology and plays a crucial role in a multitude of processes that include adhesion, migration, signaling recognition and cell-cell communication. This is accomplished by specific interactions between different membrane components such as lipids and proteins on the lipid bilayer but also through interactions with the underlying cortical actin cytoskeleton on the intracellular side and the glycocalyx matrix in close proximity to the extracellular side. Advanced biophysical techniques, including single particle tracking (SPT) have revealed that the lateral diffusion of molecular components on the plasma membrane represents a landmark manifestation of such interactions. Indeed, by studying changes in the diffusivity of individual membrane molecules, including sub-diffusion, confined diffusion and/or transient arrest of molecules in membrane compartments, it has been possible to gain insight on the nature of molecular interactions and to infer on its functional role for cell response. In this review, we will revise some exciting results where SPT has been crucial to reveal homo- and hetero-interactions on the cell membrane.

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C.

PubMed

Pagl, Roland; Aurich, Jörg E; Müller-Schlösser, Frank; Kankofer, Marta; Aurich, Christine

2006-09-15

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.

The zebrafish dystrophic mutant softy maintains muscle fibre viability despite basement membrane rupture and muscle detachment

PubMed Central

Jacoby, Arie S.; Busch-Nentwich, Elisabeth; Bryson-Richardson, Robert J.; Hall, Thomas E.; Berger, Joachim; Berger, Silke; Sonntag, Carmen; Sachs, Caroline; Geisler, Robert; Stemple, Derek L.; Currie, Peter D.

2009-01-01

Summary The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin β2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss. PMID:19736328

Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

NASA Astrophysics Data System (ADS)

Shen, Hsin-Hui; Leyton, Denisse L.; Shiota, Takuya; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

2014-10-01

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

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

Studies of molecular diffusion in single-supported bilayer lipid membranes at high hydration by quasielastic neutron scattering

NASA Astrophysics Data System (ADS)

Bai, M.; Miskowiec, A.; Wang, S.-K.; Taub, H.; Hansen, F. Y.; Jenkins, T.; Tyagi, M.; Neumann, D. A.; Diallo, S. O.; Mamontov, E.; Herwig, K. W.

2011-03-01

Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently obtained quasielastic neutron spectra from single-supported bilayer lipid membranes using the backscattering spectrometer BASIS at the Spallation Neutron Source. Protonated DMPC membranes were deposited onto Si O2 -coated Si(100) substrates and characterized by AFM. Analysis of their neutron spectra shows evidence of a relatively broad Lorentzian component that we associate with bulk-like water above a freezing temperature of ~ 267 K. At lower temperatures, the spectra differ qualitatively from that of bulk supercooled water, a behavior that we attribute to water bound to the membrane. We also find evidence of a narrow Lorentzian component that we tentatively identify with a slower motion (time scale ~ 1 ns) associated with conformational changes of the alkyl tails of the lipid molecules. Supported by NSF Grant No. DMR-0705974.

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

2016-10-01

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

PubMed Central

2011-01-01

Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work. Conclusions In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction. PMID:21241518

Cryopreservation of canine semen - new challenges.

PubMed

Farstad, W

2009-07-01

Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris-Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY-based diluents in preserving sperm survival during freezing. EY-derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which prevent cells from oxidative damage due to the generation of reactive oxygen species. An increasing number of publications now recognize the significance of protecting sperm from this damage during processing by using dietary or diluent supplemented antioxidants. This paper aims at looking at some of the new challenges in freezing of dog semen.

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

PubMed Central

Fujimoto, Toyoshi; Parmryd, Ingela

2017-01-01

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence. PMID:28119914

Interleaflet Coupling, Pinning, and Leaflet Asymmetry-Major Players in Plasma Membrane Nanodomain Formation.

PubMed

Fujimoto, Toyoshi; Parmryd, Ingela

2016-01-01

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

Fuel cells, electrolyzers, and microalgae photobioreactors: technologies for long-duration missions in human spaceflight

NASA Astrophysics Data System (ADS)

Belz, Stefan; Bretschneider, Jens; Nathanson, Emil; Buchert, Melanie

Long-duration and far-distant missions in human spaceflight have higher requirements on life support systems (LSS) technologies than for missions into low Earth orbit (LEO). LSS technologies have to ensure that humans can survive, live, and work in space. Enhancements of existing technologies, new technological developments and synergetic components integration help to close the oxygen, water and carbon loops. For these reasons, the approach of a synergetic integration of Polymer Electrolyte Membrane Fuel Cells (PEFC), Polymer Electrolyte Membrane Electrolyzers (PEL) and Photobioreactors (PBR) for microalgae cultivation into the LSS is investigated. It is demonstrated in which mission scenarii the application of PEFC, PEL, and PBR are useful in terms of mass, reliability, and cycle closures. The paper represents the current status of research at the Institute of Space Systems (IRS) of University of Stuttgart on PEFC, PEL, and PBR development. A final configuration of a prototype of a PEFC system includes the gas, water, and thermal management. The PEL is a state-of-the-art technology for space application, but the specific requirements by a synergetic integration are focused. A prototype configuration of a PBR system, which was tested under microgravity conditions in a parabolic experiment, consists of a highly sophisticated cultivation chamber, adapted sensorics, pumps, nutrients supply and harvesting unit. Additionally, the latest results of the cultivation of the microalgae species Chlorella vulgaris and Scenedesmus obliquus in the laboratories of the IRS are represented. Both species are robust, nutrient-rich for human diet. An outlook of the next steps is given for in-orbit verification.

Water Reclamation and Reuse.

PubMed

Huang, Chunkai; Zeng, Ping; Yang, Sen; Shao, Yanxi; Liu, Yang

2016-10-01

A review of the literature published in 2015 on topics relating to water reclamation and reuse is presented. The review is divided into the following sections: (1) General: extent of reuse, research needs, guidelines and monitoring, health effects; (2) Treatment technologies: integrated process design, membrane treatment, membrane bioreactors, electrocoagulation, ion exchange and adsorption, disinfection, wetlands, managed aquifer recharge; (3) Planning and management: public acceptance and education, economics/pricing, water quality planning and management and project/case studies. Much of the water treatment research focuses on membrane treatment, integrated designs, and other innovative technologies.

RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

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

Dabney-Smith, Carole

Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembledmore » proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cpTat system mechanism in chloroplasts will lead to a better understanding of the biogenesis of photosynthetic membranes potentially providing a means to engineer photosynthetic complexes into synthetic membranes for energy production. We are especially well prepared to undertake this project because we have developed a novel functional replacement assay, which was used to demonstrate a correlation of Tha4 oligomerization to transport. Thylakoids of plant chloroplasts provide a very robust, reliable assay to gain mechanistic detail about cpTat systems, providing most of the biochemical analyses to date. We plan to test our central hypothesis and accomplish the overall objective of this proposal by (1) Identifying the cpTat component(s) that interact with the mature domain of precursor during transport, (2) Determining the organization of the cpTat translocon, and (3) Comparing Tha4 topology in thylakoids during active transport and at rest. The proposed studies are innovative due to our ability to correlate structural changes in cpTat protein complexes during the transport of precursor. At the completion of this project, we expect to know the cpTat component(s) that interacts directly with the mature domain of the precursor, important because it is not known which components comprise the pore for passage of the mature domain. We also expect to know the arrangement of the components in the cpTat transport complex through direct interaction between Tha4 and the other CpTat components, a key point to establishing the mechanism of translocation. Lastly, we expect to correlate topological changes of Tha4 with precursor transport, key to establishing Tha4's role in the transport process. The successful completion of these studies is expected to have an important impact in understanding chloroplast biogenesis and assembly of photosynthetic complexes in plants and photosynthetic bacteria.« less

Electric field-induced reorganization of two-component supported bilayer membranes

PubMed Central

Groves, Jay T.; Boxer, Steven G.; McConnell, Harden M.

1997-01-01

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory’s approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi–Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes. PMID:9391034

Electric field-induced reorganization of two-component supported bilayer membranes.

PubMed

Groves, J T; Boxer, S G; McConnell, H M

1997-12-09

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

Direct Prototyping of Patterned Nanoporous Carbon: A Route from Materials to On-chip Devices

PubMed Central

Shen, Caiwei; Wang, Xiaohong; Zhang, Wenfeng; Kang, Feiyu

2013-01-01

Prototyping of nanoporous carbon membranes with three-dimensional microscale patterns is significant for integration of such multifunctional materials into various miniaturized systems. Incorporating nano material synthesis into microelectronics technology, we present a novel approach to direct prototyping of carbon membranes with highly nanoporous structures inside. Membranes with significant thicknesses (1 ~ 40 μm) are rapidly prototyped at wafer level by combining nano templating method with readily available microfabrication techniques, which include photolithography, high-temperature annealing and etching. In particular, the high-surface-area membranes are specified as three-dimensional electrodes for micro supercapacitors and show high performance compared to reported ones. Improvements in scalability, compatibility and cost make the general strategy promising for batch fabrication of operational on-chip devices or full integration of three-dimensional nanoporous membranes with existing micro systems. PMID:23887486

Membrane damage during dilution, cooling and freezing-thawing of boar spermatozoa packaged in plastic bags.

PubMed

Ortman, K; Rodriguez-Martinez, H

1994-02-01

The objective of the present investigation was to determine the degree of membrane damage in boar spermatozoa during the course of a cryopreservation procedure, including thawing. Ejaculates from four fertile Swedish Yorkshire boars were frozen under controlled conditions in Teflon-plastic bags (2.5 ml) using 3% glycerol as cryoprotectant. Membrane integrity was monitored using supravital fluorescent dyes and confirmed by scanning electron microscopy. The results show that cooling to +5 degrees C significantly affected the permeability of the plasmalemma. Supercooling (-6 degrees C) during the freezing program did not further deteriorate the integrity of the spermatozoal membrane. The thawing procedure however, dramatically increased the frequency of spermatozoa with damaged plasma membranes. Thus particular attention must be paid on designing a better thawing procedure when dealing with boar semen frozen in plastic bags.

Lipid Interaction Sites on Channels, Transporters and Receptors: Recent Insights from Molecular Dynamics Simulations

PubMed Central

Hedger, George; Sansom, Mark S. P.

2017-01-01

Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterisation of these sites is of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. PMID:26946244

Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

PubMed

Fowler, Stephanie; Akins, Mark; Bennett, Steffany A L

2016-01-01

Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.