Sample records for n-lignoceryl sphingomyelin membranes

  1. Riccardin D-N induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo

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

    Li, Lin

    Lysosomes are important targets for anticancer drug discovery. Our previous study showed that Riccardin D-N (RD-N), a natural macrocylic bisbibenzyl derivative produced by Mannich reaction, induced cell death by accumulating in lysosomes. Experiments were performed on human lung squamous cell carcinoma tissue from left inferior lobar bronchus of patient xenografts and H460 cells. RD-N was administrated for 25 days. The specimens of xenografts in Balb/c athymic (nu +/nu +) male mice were removed for immunohistochemistry, subcellular fractionation, enzyme activities and Western blotting analysis. mRFP-GFP-LC3 reporter was used to examine autophagy in H460 cells. Sphingomyelin assay was evaluated by thin-layer chromatographymore » and assay kit. Lysosomal membrane permeabilization (LMP) caused by acid sphingomyelinase (ASM) inhibition and subsequent changes of sphingomyelin (SM) metabolism selectively destabilized the cancer cell lysosomes in RD-N-treated H460 cells in vitro and tumor xenograft model in vivo. The destabilized lysosomes induced the release of cathepsins from the lysosomes into the cytosol and further triggered cell death. These results explain the underlying mechanism of RD-N induced LMP. It can be concluded that a more lysosomotropic derivative was synthesized by introduction of an amine group, which could have more potential applications in cancer therapy. - Highlights: • Riccardin D-N (RD-N) significantly downregulated LAMP1 expressions. • RD-N inhibited the acid sphingomyelinase activity. • RD-N induced lysosomal membrane permeabilization in vivo. • RD-N induced SM accumulation in the lysosomal membranes. • RD-N also induced the release of cathepsins from destabilized lysosomes.« less

  2. Anti-GM1 ganglioside antibodies modulate membrane-associated sphingomyelin metabolism by altering neutral sphingomyelinase activity.

    PubMed

    Ueda, Akihiro; Shima, Sayuri; Murate, Kenitiroh; Kikuchi, Kouichi; Nagao, Ryunosuke; Maeda, Toshiki; Muto, Eri; Niimi, Yoshiki; Mizutani, Yasuaki; Mutoh, Tatsuro

    2018-06-01

    Previous studies have shown that patients with Guillain-Barré syndrome express autoantibodies against ganglioside GM1 (GM1), although its pathogenic significance for the development of the disease remains to be elucidated. nSMase2 is the best characterized neutral sphingomyelinase (nSMase) found in neuronal cells. Activation of this enzyme leads to ceramide production, which is a known second messenger of the cell-death program in neuronal cells. We have explored the effects of anti-GM1 antibodies on sphingomyelin metabolism of PC12 cells stably transfected with human trk cDNA (PCtrk cells) by determining their effects on nSMase2 activity. The data we present here strongly suggest that anti-GM1 caused a significant change in sphingomyelin content of the membrane fraction in PCtrk cells. Both nSMase2 activity and the level of nSMase2 protein were significantly decreased by anti-GM1 treatment of PCtrk cells, while acidic SMase activities remained unchanged. Our results indicate, for the first time, that anti-GM1 may produce profound impacts on lipid metabolism in neuronal cell membranes. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Effects of cations and cholesterol with sphingomyelin membranes investigated by high-resolution broadband sum frequency vibrational spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Zhen; Feng, Rong-juan; Li, Yi-yi; Liu, Ming-hua; Guo, Yuan

    2017-08-01

    Sphingomyelin(SM) is specifically enriched in the plasma membrane of mammalian cells. Its molecular structure is compose by N-acyl-Derythro-sphingosylphosphorylcholine. The function of the SM related to membrane signaling and protein trafficking are relied on the interactions of the SM, cations, cholesterol and proteins. In this report, the interaction of three different nature SMs, cations and cholesterol at air/aqueous interfaces studied by high-resolution broadband sum frequency vibrational spectroscopy, respectively. Our results shed lights on understanding the relationship between SMs monolayer, cholesterol and Cations.

  4. Effects of beta-cyclodextrin on the structure of sphingomyelin/cholesterol model membranes.

    PubMed

    Jablin, Michael S; Flasiński, Michał; Dubey, Manish; Ratnaweera, Dilru R; Broniatowski, Marcin; Dynarowicz-Łatka, Patrycja; Majewski, Jarosław

    2010-09-08

    The interaction of beta-cyclodextrin (beta-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after beta-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by beta-CD, indicating that beta-CD is unable to remove sphingomyelin or complexed Chol. However, beta-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by beta-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of beta-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by beta-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Pathways of sphingomyelin metabolism in cultured fibroblasts from normal and sphingomyelin lipidosis subjects.

    PubMed

    Spence, M W; Clarke, J T; Cook, H W

    1983-07-25

    The metabolism of endogenous sphingomyelin labeled with 32P or [methyl-3H]choline and of exogenous [choline-methyl-3H], [32P]-, or [N-acyl-1-14C]sphingomyelin was studied in normal and Niemann-Pick Type A (NP-A) cultured fibroblasts. Despite a greater than 96% decrease in lysosomal sphingomyelinase activity in the NP-A cells, they were able to degrade endogenously produced [32P]- or [methyl-3H]sphingomyelin at normal or near normal rates. Exogenous [methyl-3H]-, [methyl-3H, 32P]-, and [methyl-3H, N-acyl-1-14C] sphingomyelin was taken up intact by normal and NP-A cells, with NP-A cells accumulating 4-8 times more lipid. By 20 h, 50% of the control cell-associated 3H and 32P was recovered in lecithin, and the ratio of activities (3H/32P) indicated most of the phosphorylcholine derived from sphingomyelin had been transferred intact. By comparison in NP-A cells, after a 40-h incubation only 20% of the labeled phosphorylcholine derived from sphingomyelin was recovered in lecithin. With both cell lines, 20 to 50 times more sphingomyelin was hydrolyzed than was taken up by the cells; the reaction products in the medium were ceramide and a mixture of water-soluble compounds such as phosphorylcholine and choline. These results indicate that there are at least two metabolic pathways for sphingomyelin modification in cultured fibroblasts in addition to degradation by the lysosomal acid sphingomyelinase. One route is hydrolysis by a cellular sphingomyelinase. The second is the hydrolysis and/or transfer of phosphorylcholine from sphingomyelin and results in the synthesis of lecithin.

  6. Development of the first sphingomyelin biomimetic stationary phase for immobilized artificial membrane (IAM) chromatography.

    PubMed

    Verzele, Dieter; Lynen, Frédéric; De Vrieze, Mike; Wright, Adrian G; Hanna-Brown, Melissa; Sandra, Pat

    2012-01-28

    A prototype sphingomyelin stationary phase for Immobilized Artificial Membrane (IAM) chromatography was synthesized by an ultra-short, solid-phase inspired methodology, in which an oxidative release monitoring strategy played a vital role. Evaluated in a proof-of-concept model for blood-brain barrier passage, partial least squares regression demonstrated its potential as an in vitro prediction tool.

  7. Staurosporines decrease ORMDL proteins and enhance sphingomyelin synthesis resulting in depletion of plasmalemmal phosphatidylserine.

    PubMed

    Maekawa, Masashi; Lee, Minhyoung; Wei, Kuiru; Ridgway, Neale D; Fairn, Gregory D

    2016-11-02

    Accumulation of phosphatidylserine in the inner leaflet of the plasma membrane is a hallmark of eukaryotes. Sublethal levels of staurosporine and related compounds deplete phosphatidylserine from the plasma membrane and abrogate K-Ras signaling. Here, we report that low-dose staurosporine and related compounds increase sphingomyelin mass. Mass-spectrometry and metabolic tracer analysis revealed an increase in both the levels and rate of synthesis of sphingomyelin in response to sublethal staurosporine. Mechanistically, it was determined that the abundance of the ORMDL proteins, which negatively regulate serine-palmitoyltransferase, are decreased by low-dose staurosporine. Finally, inhibition of ceramide synthesis, and thus sphingomyelin, prevented the displacement of phosphatidylserine and cholesterol from the inner leaflet of the plasma membrane. The results establish that an optimal level of sphingomyelin is required to maintain the distribution of phosphatidylserine and cholesterol in the plasma membrane and further demonstrate a complex relationship between the trafficking of phosphatidylserine and sphingomyelin.

  8. Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.

    PubMed Central

    Pörn, M I; Slotte, J P

    1990-01-01

    Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin. PMID:2222406

  9. Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs

    PubMed Central

    Kinoshita, Masanao; Suzuki, Kenichi G.N.; Takada, Misa; Ano, Hikaru; Abe, Mitsuhiro; Makino, Asami; Kobayashi, Toshihide; Hirosawa, Koichiro M.; Fujiwara, Takahiro K.; Murata, Michio

    2017-01-01

    Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM. PMID:28330937

  10. Pressure-temperature phase behavior of mixtures of natural sphingomyelin and ceramide extracts.

    PubMed

    Barriga, Hanna M G; Parsons, Edward S; McCarthy, Nicola L C; Ces, Oscar; Seddon, John M; Law, Robert V; Brooks, Nicholas J

    2015-03-31

    Ceramides are a group of sphingolipids that act as highly important signaling molecules in a variety of cellular processes including differentiation and apoptosis. The predominant in vivo synthetic pathway for ceramide formation is via sphingomyelinase catalyzed hydrolysis of sphingomyelin. The biochemistry of this essential pathway has been studied in detail; however, there is currently a lack of information on the structural behavior of sphingomyelin- and ceramide-rich model membrane systems, which is essential for developing a bottom-up understanding of ceramide signaling and platform formation. We have studied the lyotropic phase behavior of sphingomyelin-ceramide mixtures in excess water as a function of temperature (30-70 °C) and pressure (1-200 MPa) by small- and wide-angle X-ray scattering. At low ceramide concentrations the mixtures form the ripple gel phase (P(β)') below the gel transition temperature for sphingomyelin, and this observation has been confirmed by atomic force microscopy. Formation of the ripple gel phase can also be induced at higher temperatures via the application of hydrostatic pressure. At high ceramide concentration an inverse hexagonal phase (HII) is formed coexisting with a cubic phase.

  11. Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

    PubMed

    Pérez-Cañamás, A; Benvegnù, S; Rueda, C B; Rábano, A; Satrústegui, J; Ledesma, M D

    2017-05-01

    Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

  12. Interaction between sphingomyelin and oxysterols contributes to atherosclerosis and sudden death

    PubMed Central

    Kummerow, Fred A

    2013-01-01

    Despite major public health efforts, coronary heart disease continues to be the leading cause of death in the United States. Oxidized lipids contribute to heart disease both by increasing deposition of calcium on the arterial wall, a major hallmark of atherosclerosis, and by interrupting blood flow, a major contributor to heart attack and sudden death. Oxidized cholesterol (oxysterols) enhances the production of sphingomyelin, a phospholipid found in the cellular membranes of the coronary artery. This increases the sphingomyelin content in the cell membrane, which in turn enhances the interaction between the membrane and ionic calcium (Ca2+), thereby increasing the risk of arterial calcification. Patients undergoing bypass surgery had greater concentrations of oxysterols in their plasma than cardiac catheterized controls with no stenosis, and had five times more sphingomyelin in their arteries than in the artery of the placenta of a newborn. The oxysterols found in the plasma of these patients were also found in the plasma of rabbits that had been fed oxidized cholesterol and in frying fats and powdered egg yolk intended for human consumption. Together these findings suggest that oxysterols found in the diet are absorbed and contribute to arterial calcification. Oxidized low-density lipoprotein (OxLDL) further contributes to heart disease by increasing the synthesis of thromboxane in platelets, which increases blood clotting. Cigarette smoke and trans fatty acids, found in partially hydrogenated soybean oil, both inhibit the synthesis of prostacyclin, which inhibits blood clotting. By increasing the ratio of thromboxane to prostacyclin, these factors interact to interrupt blood flow, thereby contributing to heart attack and sudden death. Levels of oxysterols and OxLDL increase primarily as a result of three diet or lifestyle factors: the consumption of oxysterols from commercially fried foods such as fried chicken, fish, and french fries; oxidation of cholesterol

  13. Cloning, characterization, expression and comparative analysis of pig Golgi membrane sphingomyelin synthase 1.

    PubMed

    Guillén, Natalia; Navarro, María A; Surra, Joaquín C; Arnal, Carmen; Fernández-Juan, Marta; Cebrián-Pérez, Jose Alvaro; Osada, Jesús

    2007-02-15

    Pig sphingomyelin synthase 1 (SMS1) cDNA was cloned, characterized and compared to the human ortholog. Porcine protein consists of 413 amino acids and displays a 97% sequence identity with human protein. A phylogenic tree of proteins reveals that porcine SMS1 is more closely related to bovine and rodent proteins than to human. Analysis of protein mass was higher than the theoretical prediction based on amino acid sequence suggesting a kind of posttranslational modification. Quantitative representation of tissue distribution obtained by real-time RT-PCR showed that it was widely expressed although important variations in levels were obtained among organs. Thus, the cardiovascular system, especially the heart, showed the highest value of all the tissues studied. Regional differences of expression were observed in the central nervous system and intestinal tract. Analysis of the hepatic mRNA and protein expressions of SMS1 following turpentine treatment revealed a progressive decrease in the former paralleled by a decrease in the protein concentration. These findings indicate the variation in expression in the different tissues might suggest a different requirement of Golgi sphingomyelin for the specific function in each organ and a regulation of the enzyme in response to turpentine-induced hepatic injury.

  14. Effects of bile salts on glucosylceramide containing membranes.

    PubMed

    Halin, Josefin; Mattjus, Peter

    2011-12-01

    The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

    PubMed Central

    Gensure, Rebekah H.; Zeidel, Mark L.; Hill, Warren G.

    2006-01-01

    H+/OH− permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH− and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH− permeability from a baseline of 2.4×10−2 cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH− permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH− permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484–1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH− conductivity, (iii) H+/OH− fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH− permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results. PMID

  16. Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content.

    PubMed

    Abi-Rizk, Georges; Besson, Françoise

    2008-10-15

    The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.

  17. Sphingomyelins and ceramides with VLCPUFAs are excluded from low-density raft-like domains in differentiating spermatogenic cells[S

    PubMed Central

    Santiago Valtierra, Florencia X.; Mateos, Melina V.; Aveldaño, Marta I.; Oresti, Gerardo M.

    2017-01-01

    Rat spermatogenic cells contain sphingomyelins (SMs) and ceramides (Cers) with very long-chain PUFAs (VLCPUFAs) in nonhydroxylated (n-V) and 2-hydroxylated (h-V) forms. How these atypical species distribute among membrane fractions during differentiation was investigated here using a detergent-free procedure to isolate a small light raft-like low-density fraction and a large heavy fraction, mostly derived from the plasma membrane of spermatocytes, round spermatids, and late spermatids. The light fraction contained cholesterol, glycerophospholipids (GPLs), and SM with the same saturated fatty acids in all three stages. In the heavy fraction, as PUFA increased in the GPL and VLCPUFA in SM from spermatocytes to spermatids, the concentration of cholesterol was also augmented. The heavy fraction had mostly n-V SM in spermatocytes, but accumulated h-V SM and h-V Cer in spermatids. A fraction containing intracellular membranes had less SM and more Cer than the latter, but in both fractions SM and Cer species with h-V increased over species with n-V with differentiation. This accretion of h-V was consistent with the differentiation-dependent expression of fatty acid 2-hydroxylase (Fa2h), as it increased significantly from spermatocytes to spermatids. The non-raft region of the plasma membrane is thus the main target of the dynamic lipid synthesis and remodeling that is involved in germ cell differentiation. PMID:28082410

  18. Sphingomyelin metabolism controls the shape and function of the Golgi cisternae

    PubMed Central

    Campelo, Felix; van Galen, Josse; Turacchio, Gabriele; Parashuraman, Seetharaman; Kozlov, Michael M; García-Parajo, María F; Malhotra, Vivek

    2017-01-01

    The flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae. DOI: http://dx.doi.org/10.7554/eLife.24603.001 PMID:28500756

  19. 21 CFR 862.1455 - Lecithin/sphingomyelin ratio in amniotic fluid test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lecithin/sphingomyelin ratio in amniotic fluid... Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a) Identification. A lecithin/sphingomyelin ratio in amniotic fluid test system is a device intended to measure the...

  20. Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

    PubMed

    Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

    2013-11-07

    Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

  1. Enhancement of proton transfer in ion channels by membrane phosphate headgroups.

    PubMed

    Wyatt, Debra L; de Godoy, Carlos Marcelo G; Cukierman, Samuel

    2009-05-14

    The transfer of protons (H+) in gramicidin (gA) channels is markedly distinct in monoglyceride and phospholipid membranes. In this study, the molecular groups that account for those differences were investigated using a new methodology. The rates of H+ transfer were measured in single gA channels reconstituted in membranes made of plain ceramides or sphingomyelins and compared to those in monoglyceride and phospholipid bilayers. Single-channel conductances to protons (gH) were significantly larger in sphingomyelin than in ceramide membranes. A novel and unsuspected finding was that H+ transfer was heavily attenuated or completely blocked in ceramide (but not in sphingomyelin) membranes in low-ionic-strength solutions. It is reasoned that H-bond dynamics at low ionic strengths between membrane ceramides and gA makes channels dysfunctional. The rate of H+ transfer in gA channels in ceramide membranes is significantly higher than that in monoglyceride bilayers. This suggests that solvation of the hydrophobic surface of gA channels by two acyl chains in ceramides stabilizes the gA channels and the water wire inside the pore, leading to an enhancement of H+ transfer in relation to that occurring in monoglyceride membranes. gH values in gA channels are similar in ceramide and monoglyceride bilayers and in sphingomyelin and phospholipid membranes. It is concluded that phospho headgroups in membranes have significant effects on the rate of H+ transfer at the membrane gA channel/solution interfaces, enhancing the entry and exit rates of protons in channels.

  2. [Characteristics of fatty acid composition of phosphatidyl cholines and sphingomyelins of low-density lipoproteins in the plasma of native inhabitants of Chukotka].

    PubMed

    Gerasimova, E N; Levachev, M M; Perova, N V; Nikitin, Iu P; Ozerova, I N

    1986-01-01

    Contents of cholesterol, triglycerides, high density lipoproteins (HDL) cholesterol as well as phospholipid and fatty acid compositions of phosphatidyl cholines and sphingomyelins in low density lipoproteins (LDL) were studied in blood plasma of Chukot aborigenes--Eskimos as compared with Moscow inhabitants. In Eskimos content of HDL cholesterol was higher but concentration of cholesterol and triglycerides was lower in blood plasma. In LDL concentration of sphingomyelins was increased and fatty acid composition of phosphatidyl cholines and sphingomyelins was altered where amount of polyunsaturated fatty acids was elevated (20:5 + 22:5 + 22:6). The specific characteristics of the LDL phospholipids observed in Eskimos might be responsible for the higher liquid properties of the surface monolayer in the lipoproteins; this alteration might be important for the lipoprotein properties and transformation as well as for the properties of membrane-bound enzymes, for synthesis of thromboxane and prostacyclins.

  3. Optimization of a Histopathological Biomarker for Sphingomyelin Accumulation in Acid Sphingomyelinase Deficiency

    PubMed Central

    Johnson, Jennifer; Maloney, Colleen L.; Yandl, Emily; Griffiths, Denise; Thurberg, Beth L.; Ryan, Susan

    2012-01-01

    Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy. PMID:22614361

  4. Effects of short peptides on thymocyte blast transformation and signal transduction along the sphingomyelin pathway.

    PubMed

    Khavinson, V Kh; Rybakina, E G; Malinin, V V; Pivanovich, I Yu; Shanin, S N; Korneva, E A

    2002-05-01

    Immunomodulating effects of synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) and possible involvement of the sphingomyelin signal transduction pathway in their effects in mouse thymocytes were studied. Vilon produced the most potent comitogenic effect on thymocyte proliferation and modulated comitogenic activity of interleukin-1b. Epithalon was less potent, while Cortagen produced no such effects. Vilon produced a more pronounced stimulatory effect on sphingomyelinase activity in mouse thymocyte membranes compared to Epithalon and Cortagen.

  5. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

    PubMed

    van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

    2002-06-21

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

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

    PubMed

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

    2012-01-01

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

  7. Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes.

    PubMed

    Pankov, R; Markovska, T; Antonov, P; Ivanova, L; Momchilova, A

    2006-09-01

    Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers.

  8. 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.

  9. Phosphatidylinositol transfer protein beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin.

    PubMed

    Ségui, Bruno; Allen-Baume, Victoria; Cockcroft, Shamshad

    2002-08-15

    Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater. To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment. To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously. However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.

  10. Automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid.

    PubMed

    Bradley, C A; Salhany, K E; Entman, S S; Aleshire, S L; Parl, F F

    1987-01-01

    We describe methods for automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid. Phospholipase C (EC 3.1.4.3) and sphingomyelin phosphodiesterase (EC 3.1.4.12) are reacted with lecithin and sphingomyelin, respectively, to liberate phosphocholine. Phosphocholine is then reacted with alkaline phosphatase, choline oxidase, peroxidase, and 4-aminoantipyrine to form a colored complex, for which the absorbance at 500 nm is measured with a centrifugal analyzer. Phosphatidylglycerol is hydrolyzed by phospholipase D (EC 3.1.4.4) to form glycerol, which is subsequently reacted with ATP and NAD+ in the presence of glycerol kinase and glycerol-3-phosphate dehydrogenase to yield NADH. The absorbance of the NADH formed is measured at 340 nm. These methods provide a simple, rapid, and accurate alternative to thin-layer chromatography for determination of phospholipids in amniotic fluid for assessment of fetal lung maturity.

  11. Structure of Sphingomyelin Bilayers: A Simulation Study

    PubMed Central

    Chiu, S. W.; Vasudevan, S.; Jakobsson, Eric; Mashl, R. Jay; Scott, H. Larry

    2003-01-01

    We have carried out a molecular dynamics simulation of a hydrated 18:0 sphingomyelin lipid bilayer. The bilayer contained 1600 sphingomyelin (SM) molecules, and 50,592 water molecules. After construction and initial equilibration, the simulation was run for 3.8 ns at a constant temperature of 50°C and a constant pressure of 1 atm. We present properties of the bilayer calculated from the simulation, and compare with experimental data and with properties of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The SM bilayers are significantly more ordered and compact than DPPC bilayers at the same temperature. SM bilayers also exhibit significant intramolecular hydrogen bonding between phosphate ester oxygen and hydroxyl hydrogen atoms. This results in a decreased hydration in the polar region of the SM bilayer compared with DPPC. Since our simulation system is very large we have calculated the power spectrum of bilayer undulation and peristaltic modes, and we compare these data with similar calculations for DPPC bilayers. We find that the SM bilayer has significantly larger bending modulus and area compressibility compared to DPPC. PMID:14645055

  12. Plasma phosphatidylcholine and sphingomyelin concentrations are associated with depression and anxiety symptoms in a Dutch family-based lipidomics study.

    PubMed

    Demirkan, Ayşe; Isaacs, Aaron; Ugocsai, Peter; Liebisch, Gerhard; Struchalin, Maksim; Rudan, Igor; Wilson, James F; Pramstaller, Peter P; Gyllensten, Ulf; Campbell, Harry; Schmitz, Gerd; Oostra, Ben A; van Duijn, Cornelia M

    2013-03-01

    The central nervous system has the second highest concentration of lipids after adipose tissue. Alterations in neural membrane phospho- and sphingolipid composition can influence crucial intra- and intercellular signalling and alter the membrane's properties. Recently, the polyunsaturated fatty acids (PUFA) hypothesis for depression suggests that phospho- and sphingolipid metabolism includes potential pathways for the disease. In 742 people from a Dutch family-based study, we assessed the relationships between 148 different plasma phospho- and sphingolipid species and depression/anxiety symptoms as measured by the Hospital Anxiety and Depression Scales (HADS-A and HADS-D) and the Centre for Epidemiological Studies Depression Scale (CES-D). We observed significant differences in plasma sphingomyelins (SPM), particularly the SPM 23:1/SPM 16:0 ratio, which was inversely correlated with depressive symptom scores. We observed a similar trend for plasma phosphatidylcholines (PC), particularly the molar proportion of PC O 36:4 and its ratio to ceramide CER 20:0. Absolute levels of PC O 36:4 were also associated with depression symptoms in an independent replication. To our knowledge this is the first study on depressive symptoms that focuses on specific phospho- and sphingolipid molecules in plasma rather than total PUFA concentrations. The findings of this lipidomic study suggests that plasma sphingomyelins and ether phospholipids should be further studied for their potential as biomarkers and for a better understanding of the underlying mechanisms of this systemic disease. Copyright © 2012. Published by Elsevier Ltd.

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

    PubMed

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

    2015-12-01

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

  14. Novel Interconnections in Lipid Metabolism Revealed by Overexpression of Sphingomyelin Synthase-1*

    PubMed Central

    Deevska, Gergana M.; Dotson, Patrick P.; Karakashian, Alexander A.; Isaac, Giorgis; Wrona, Mark; Kelly, Samuel B.; Merrill, Alfred H.; Nikolova-Karakashian, Mariana N.

    2017-01-01

    This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids. PMID:28087695

  15. Interaction of cholesterol with sphingomyelins and acyl-chain-matched phosphatidylcholines: a comparative study of the effect of the chain length.

    PubMed Central

    Ramstedt, B; Slotte, J P

    1999-01-01

    In this study we have synthesized sphingomyelins (SM) and phosphatidylcholines (PC) with amide-linked or sn-2 linked acyl chains with lengths from 14 to 24 carbons. The purpose was to examine how the chain length and degree of unsaturation affected the interaction of cholesterol with these phospholipids in model membrane systems. Monolayers of saturated SMs and PCs with acyl chain lengths above 14 carbons were condensed and displayed a high collapse pressure ( approximately 70 mN/m). Monolayers of N-14:0-SM and 1(16:0)-2(14:0)-PC had a much lower collapse pressure (58-60 mN/m) and monounsaturated SMs collapsed at approximately 50 mN/m. The relative interaction of cholesterol with these phospholipids was determined at 22 degreesC by measuring the rate of cholesterol desorption from mixed monolayers (50 mol % cholesterol; 20 mN/m) to beta-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated SM monolayers than from chain-matched PC monolayers. In SM monolayers, the rate of cholesterol desorption was very slow for all N-linked chains, whereas for PC monolayers we could observe higher desorption rates from monolayers of longer PCs. These results show that cholesterol interacts favorably with SMs (low rate of desorption), whereas its interaction (or miscibility) with long chain PCs is weaker. Introduction of a single cis-unsaturation in the N-linked acyl chain of SMs led to faster rates of cholesterol desorption as compared with saturated SMs. The exception was monolayers of N-22:1-SM and N-24:1-SM from which cholesterol desorbed almost as slowly as from the corresponding saturated SM monolayers. The results of this study suggest that cholesterol is most likely capable of interacting with all physiologically relevant (including long-chain) SMs present in the plasma membrane of cells. PMID:9929492

  16. Cholesterol blocks spontaneous insertion of membrane proteins into liposomes of phosphatidylcholine.

    PubMed

    Nakamura, Shota; Suzuki, Sonomi; Saito, Hiroaki; Nishiyama, Ken-Ichi

    2018-04-01

    Spontaneous insertion of membrane proteins into liposomes formed from Escherichia coli polar phospholipids is blocked by diacylglycerol (DAG) at a physiological level. We found that cholesterol also blocks this spontaneous insertion, although a much larger amount is necessary for sufficient blockage. Reversely, sphingomyelin enhanced the spontaneous insertion. DAG at a physiological level was found not to block spontaneous insertion into liposomes formed from phosphatidylcholine (PC), while non-physiologically high concentrations of DAG reduced it. On the other hand, cholesterol blocked the spontaneous insertion into PC liposomes at a physiological level, explaining that both PC and cholesterol are absent in E. coli. While sphingomyelin did not enhance spontaneous insertion into PC liposomes, the effect of cholesterol on blockage of spontaneous insertion was dominant over that of sphingomyelin, suggesting that cholesterol functions as a blocker of disordered spontaneous insertion in eukaryotic cells. Lower amount of cholesterol was necessary to block spontaneous insertion into ER-mimic liposomes, explaining that ER membranes contain less amount of cholesterol. These results also explain that cholesterol, but not DAG, is involved in blockage of spontaneous insertion in eukaryotic cells, since DAG plays an important role as a second messenger in signal transduction.

  17. Nanoscale Packing Differences in Sphingomyelin and Phosphatidylcholine Revealed by BODIPY Fluorescence in Monolayers: Physiological Implications

    PubMed Central

    2015-01-01

    Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force–area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force–area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match. PMID:24564829

  18. Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma.

    PubMed

    Piehl, Lidia L; Cisale, Humberto; Torres, Natalia; Capani, Francisco; Sterin-Speziale, Norma; Hager, Alfredo

    2006-05-01

    Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine

  19. Trivalent chromium induces autophagy by activating sphingomyelin phosphodiesterase 2 and increasing cellular ceramide levels in renal HK2 cells.

    PubMed

    Yang, Cheng-Lin; Chiou, Shiow-Her; Tai, Wei-Chun; Joseph, Nithila A; Chow, Kuan-Chih

    2017-11-01

    In this study, we examined the role of autophagy in the initiation of lipid increases in renal epithelial HK2 cells. We found that trivalent chromium [Cr(III)] induced autophagy by activating sphingomyelin phosphodiesterase 2 (SMPD2). SMPD2 increases levels of ceramide and other lipids. Confocal immunofluorescence microscopy showed that signals of ceramide overlapped with LC3, suggesting that ceramide might play an important role in the formation of autophagosome. In conclusion, our data indicate that Cr(III) induces autophagy via structural aberration of organelle membrane, in particular by the increase of lipid compositions in addition to autophagy-associated proteins. © 2017 Wiley Periodicals, Inc.

  20. Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking*

    PubMed Central

    Lima, Santiago; Milstien, Sheldon; Spiegel, Sarah

    2017-01-01

    The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking. PMID:28049734

  1. Lysosomal degradation of membrane lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2010-05-03

    The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Phospholipid lateral diffusion in phosphatidylcholine-sphingomyelin-cholesterol monolayers; effects of oxidatively truncated phosphatidylcholines.

    PubMed

    Parkkila, Petteri; Stefl, Martin; Olżyńska, Agnieszka; Hof, Martin; Kinnunen, Paavo K J

    2015-01-01

    Oxidative stress is involved in a number of pathological conditions and the generated oxidatively modified lipids influence membrane properties and functions, including lipid-protein interactions and cellular signaling. Brewster angle microscopy demonstrated oxidatively truncated phosphatidylcholines to promote phase separation in monolayers of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). More specifically, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), was found to increase the miscibility transition pressure of the SM/Chol-phase. Lateral diffusion of lipids is influenced by a variety of membrane properties, thus making it a sensitive parameter to observe the coexistence of different lipid phases, for instance. The dependence on lipid lateral packing of the lateral diffusion of fluorophore-containing phospholipid analogs was investigated in Langmuir monolayers composed of POPC, SM, and Chol and additionally containing oxidatively truncated phosphatidylcholines, using fluorescence correlation spectroscopy (FCS). To our knowledge, these are the first FCS results on miscibility transition in ternary lipid monolayers, confirming previous results obtained using Brewster angle microscopy on such lipid monolayers. Wide-field fluorescence microscopy was additionally employed to verify the transition, i.e. the loss and reformation of SM/Chol domains. Copyright © 2014. Published by Elsevier B.V.

  3. 21 CFR 862.1455 - Lecithin/sphingomyelin ratio in amniotic fluid test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a...

  4. Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice

    PubMed Central

    Scherthan, Harry; Horsch, Marion; Beckers, Johannes; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Ford, Steven J.; Burton, Neal C.; Razansky, Daniel; Trümbach, Dietrich; Aichler, Michaela; Walch, Axel Karl; Calzada-Wack, Julia; Neff, Frauke; Wurst, Wolfgang; Hartmann, Tobias; Floss, Thomas

    2016-01-01

    Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility. PMID:27788151

  5. Lateral microheterogeneity of diphenylhexatriene-labeled choline phospholipids in the erythrocyte ghost membrane as determined by time-resolved fluorescence spectroscopy.

    PubMed

    Prenner, E; Sommer, A; Maurer, N; Glatter, O; Gorges, R; Paltauf, F; Hermetter, A

    2000-04-01

    Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.

  6. A study of the lecithin/sphingomyelin ratio of amniotic fluid.

    PubMed

    Gebhardt, D O; Beintema, A; de Rooij, R E; Wildeboer, F N; Merkus, J M

    1975-10-15

    1. There is a significant correlation between lecithin/sphingomyelin (L/S) ratios based on densitometry (L/S)D and L/S ratios based on phosphorus determinations ((L/S)P). 2. The fetal lung is mature when the (L/S)D, determined according to Verhoeven, A.G.J. and Merkus, H.M.W.M. (1974) Clin. Chim. Acta 53, 229--232, is 1.2. This value is equivalent to an (L/S)P of 1.8. 3. The acetone precipitation procedure, introduced by Gluck, L., Kulovich, M.V., Borer, R.C. and Keidel, W.N. (1974) Am. J. Obstet. Gynecol. 120, 142--155, is a necessary step for isolating surface-active lecithin. 4. Standardization of the (L/S)D test is feasible and should permit different laboratories to use the same transition point.

  7. Lateral Membrane Heterogeneity Regulates Viral-Induced Membrane Fusion during HIV Entry

    PubMed Central

    Molotkovsky, Rodion J.; Alexandrova, Veronika V.; Galimzyanov, Timur R.; Jiménez-Munguía, Irene; Pavlov, Konstantin V.; Akimov, Sergey A.

    2018-01-01

    Sphingomyelin- and cholesterol- enriched membrane domains, commonly referred to as “rafts” play a crucial role in a large number of intra- and intercellular processes. Recent experiments suggest that not only the volumetric inhomogeneity of lipid distribution in rafts, but also the arrangement of the 1D boundary between the raft and the surrounding membrane is important for the membrane-associated processes. The reason is that the boundary preferentially recruits different peptides, such as HIV (human immunodeficiency virus) fusion peptide. In the present work, we report a theoretical investigation of mechanisms of influence of the raft boundary arrangement upon virus-induced membrane fusion. We theoretically predict that the raft boundary can act as an attractor for viral fusion peptides, which preferentially distribute into the vicinity of the boundary, playing the role of ‘line active components’ of the membrane (‘linactants’). We have calculated the height of the fusion energy barrier and demonstrated that, in the case of fusion between HIV membrane and the target cell, presence of the raft boundary in the vicinity of the fusion site facilitates fusion. The results we obtained can be further generalized to be applicable to other enveloped viruses. PMID:29772704

  8. Lateral Membrane Heterogeneity Regulates Viral-Induced Membrane Fusion during HIV Entry.

    PubMed

    Molotkovsky, Rodion J; Alexandrova, Veronika V; Galimzyanov, Timur R; Jiménez-Munguía, Irene; Pavlov, Konstantin V; Batishchev, Oleg V; Akimov, Sergey A

    2018-05-16

    Sphingomyelin- and cholesterol- enriched membrane domains, commonly referred to as "rafts" play a crucial role in a large number of intra- and intercellular processes. Recent experiments suggest that not only the volumetric inhomogeneity of lipid distribution in rafts, but also the arrangement of the 1D boundary between the raft and the surrounding membrane is important for the membrane-associated processes. The reason is that the boundary preferentially recruits different peptides, such as HIV (human immunodeficiency virus) fusion peptide. In the present work, we report a theoretical investigation of mechanisms of influence of the raft boundary arrangement upon virus-induced membrane fusion. We theoretically predict that the raft boundary can act as an attractor for viral fusion peptides, which preferentially distribute into the vicinity of the boundary, playing the role of 'line active components' of the membrane ('linactants'). We have calculated the height of the fusion energy barrier and demonstrated that, in the case of fusion between HIV membrane and the target cell, presence of the raft boundary in the vicinity of the fusion site facilitates fusion. The results we obtained can be further generalized to be applicable to other enveloped viruses.

  9. Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation.

    PubMed

    Christomanou, H

    1980-10-01

    1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.

  10. [PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

    PubMed

    Sudarkina, O Yu; Dergunova, L V

    2015-01-01

    Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

  11. Behavior of sphingomyelin and ceramide in a tear film lipid layer model.

    PubMed

    Olżyńska, Agnieszka; Cwiklik, Lukasz

    2017-03-01

    Tear film lipid layer is a complex lipid mixture forming the outermost interface between eye and environment. Its key characteristics, such as surface tension and structural stability, are governed by the presence of polar lipids. The origin of these lipids and exact composition of the mixture are still elusive. We focus on two minor polar lipid components of the tear film lipid later: sphingomyelin and ceramide. By employing coarse grain molecular dynamics in silico simulations accompanied by Langmuir balance experiments we provide molecular-level insight into behavior of these two lipids in a tear film lipid layer model. Sphingomyelin headgroups are significantly exposed at the water-lipids boundary while ceramide molecules are incorporated between other lipids frequently interacting with nonpolar lipids. Even though these two lipids increase surface tension of the film, their molecular-level behavior suggests that they have a stabilizing effect on the tear film lipid layer. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. Limonoid Compounds Inhibit Sphingomyelin Biosynthesis by Preventing CERT Protein-dependent Extraction of Ceramides from the Endoplasmic Reticulum*

    PubMed Central

    Hullin-Matsuda, Françoise; Tomishige, Nario; Sakai, Shota; Ishitsuka, Reiko; Ishii, Kumiko; Makino, Asami; Greimel, Peter; Abe, Mitsuhiro; Laviad, Elad L.; Lagarde, Michel; Vidal, Hubert; Saito, Tamio; Osada, Hiroyuki; Hanada, Kentaro; Futerman, Anthony H.; Kobayashi, Toshihide

    2012-01-01

    To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism. PMID:22605339

  13. Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

    PubMed

    Sun, Bingyun; Hood, Leroy

    2014-06-06

    The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

  14. Invariant natural killer T cells from children with versus without food allergy exhibit differential responsiveness to milk-derived sphingomyelin.

    PubMed

    Jyonouchi, Soma; Abraham, Valsamma; Orange, Jordan S; Spergel, Jonathan M; Gober, Laura; Dudek, Emily; Saltzman, Rushani; Nichols, Kim E; Cianferoni, Antonella

    2011-07-01

    A key immunologic feature of food allergy (FA) is the presence of a T(h)2-type cytokine bias. Ligation of the invariant natural killer T cell (iNKT) T-cell receptor (TCR) by sphingolipids presented via the CD1d molecule leads to copious secretion of T(h)2-type cytokines. Major food allergens (eg, milk, egg) are the richest dietary source of sphingolipids (food-derived sphingolipids [food-SLs]). Nonetheless, the role of iNKTs in FA is unknown. To investigate the role of iNKTs in FA and to assess whether food-SL-CD1d complexes can engage the iNKT-TCR and induce iNKT functions. PBMCs from 15 children with cow's milk allergy (MA), 12 children tolerant to cow's milk but with allergy to egg, and 13 healthy controls were incubated with α-galactosylceramide (αGal), cow's milk-sphingomyelin, or hen's egg-ceramide. iNKTs were quantified, and their cytokine production and proliferation were assessed. Human CD1d tetramers loaded with milk-sphingomyelin or egg-ceramide were used to determine food-SL binding to the iNKT-TCR. Milk-sphingomyelin, but not egg-ceramide, can engage the iNKT-TCR and induce iNKT proliferation and T(h)2-type cytokine secretion. Children with FA, especially those with MA, had significantly fewer peripheral blood iNKTs and their iNKTs exhibited a greater T(h)2 response to αGal and milk-sphingomyelin than iNKTs of healthy controls. iNKTs from children with FA, especially those with MA, are reduced in number and exhibit a T(h)2 bias in response to αGal and milk-sphingomyelin. These data suggest a potential role for iNKTs in FA. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  15. Impact of amphiphilic molecules on the structure and stability of homogeneous sphingomyelin bilayer: Insights from atomistic simulations

    NASA Astrophysics Data System (ADS)

    Kumari, Pratibha; Kaur, Supreet; Sharma, Shobha; Kashyap, Hemant K.

    2018-04-01

    Modulation of lipid membrane properties due to the permeation of amphiphiles is an important biological process pertaining to many applications in the field of pharmaceutics, toxicology, and biotechnology. Sphingolipids are both structural and functional lipids that constitute an important component of mechanically stable and chemically resistant outer leaflets of plasma membranes. Here, we present an atomistic molecular dynamics simulation study to appreciate the concentration-dependent effects of small amphiphilic molecules, such as ethanol, acetone, and dimethyl sulfoxide (DMSO), on the structure and stability of a fully hydrated homogeneous N-palmitoyl-sphingomyelin (PSM) bilayer. The study reveals an increase in the lateral expansion of the bilayer along with disordering of the hydrophobic lipid tails on increasing the concentration of ethanol. At higher concentrations of ethanol, rupturing of the bilayer is quite evident through the analysis of partial electron density profiles and lipid tail order parameters. For ethanol containing systems, permeation of water molecules in the hydrophobic part of the bilayer is allowed through local defects made due to the entry of ethanol molecules via ethanol-ethanol and ethanol-PSM hydrogen bonds. Moreover, the extent of PSM-PSM hydrogen bonding decreases with increasing ethanol concentration. On the other hand, acetone and DMSO exhibit minimal effects on the stability of the PSM bilayer at their lower concentrations, but at higher concentrations they tend to enhance the stability of the bilayer. The simulated potential of mean force (PMF) profiles for the translocation of the three solutes studied reveal that the free-energy of transfer of an ethanol molecule across the PSM lipid head region is lower than that for acetone and DMSO molecules. However, highest free-energy rise in the core hydrophobic part of the bilayer is observed for the DMSO molecule, whereas the ethanol and acetone PMF profiles show a lower barrier in

  16. A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia.

    PubMed

    Ermini, Leonardo; Ausman, Jonathan; Melland-Smith, Megan; Yeganeh, Behzad; Rolfo, Alessandro; Litvack, Michael L; Todros, Tullia; Letarte, Michelle; Post, Martin; Caniggia, Isabella

    2017-09-22

    Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women.

  17. Microwave Energy Increases Fatty Acid Methyl Ester Yield in Human Whole Blood Due to Increased Sphingomyelin Transesterification.

    PubMed

    Metherel, Adam H; Aristizabal Henao, Juan J; Ciobanu, Flaviu; Taha, Ameer Y; Stark, Ken D

    2015-09-01

    Dried blood spots (DBS) by fingertip prick collection for fatty acid profiling are becoming increasingly popular due to ease of collection, minimal invasiveness and its amenability to high-throughput analyses. Herein, we assess a microwave-assisted direct transesterification method for the production of fatty acid methyl esters (FAME) from DBS. Technical replicates of human whole blood were collected and 25-μL aliquots were applied to chromatography strips prior to analysis by a standard 3-h transesterification method or microwave-assisted direct transesterification method under various power (variable vs constant), time (1-5 min) and reagent (1-10% H2SO4 in methanol) conditions. In addition, a standard method was compared to a 5-min, 30-W power microwave in 1% H2SO4 method for FAME yield from whole blood sphingomyelin, and sphingomyelin standards alone and spiked in whole blood. Microwave-assisted direct transesterification yielded no significant differences in both quantitative (nmol/100 µL) and qualitative (mol%) fatty acid assessments after as little as 1.5- and 1-min reaction times, respectively, using the variable power method and 5% H2SO4 in methanol. However, 30-W power for 5 min increased total FAME yield of the technical replicates by 14%. This increase appears largely due to higher sphingomyelin-derived FAME yield of up to 109 and 399% compared to the standard method when determined from whole blood or pure standards, respectively. In conclusion, microwave-assisted direct transesterification of DBS achieved in as little as 1-min, and 5-min reaction times increase total fatty acids primarily by significantly improving sphingomyelin-derived fatty acid yield.

  18. Membrane lipidomics in schizophrenia patients: a correlational study with clinical and cognitive manifestations.

    PubMed

    Tessier, C; Sweers, K; Frajerman, A; Bergaoui, H; Ferreri, F; Delva, C; Lapidus, N; Lamaziere, A; Roiser, J P; De Hert, M; Nuss, P

    2016-10-04

    Schizophrenia is a severe mental condition in which several lipid abnormalities-either structural or metabolic-have been described. We tested the hypothesis that an abnormality in membrane lipid composition may contribute to aberrant dopamine signaling, and thereby symptoms and cognitive impairment, in schizophrenia (SCZ) patients. Antipsychotic-medicated and clinically stable SCZ outpatients (n=74) were compared with matched healthy subjects (HC, n=40). A lipidomic analysis was performed in red blood cell (RBC) membranes examining the major phospholipid (PL) classes and their associated fatty acids (FAs). Clinical manifestations were examined using the positive and negative syndrome scale (PANSS). Cognitive function was assessed using the Continuous Performance Test, Salience Attribution Test and Wisconsin Card Sorting Test. Sphingomyelin (SM) percentage was the lipid abnormality most robustly associated with a schizophrenia diagnosis. Two groups of patients were defined. The first group (SCZ c/SM-) is characterized by a low SM membrane content. In this group, all other PL classes, plasmalogen and key polyunsaturated FAs known to be involved in brain function, were significantly modified, identifying a very specific membrane lipid cluster. The second patient group (SCZ c/SM+) was similar to HCs in terms of RBC membrane SM composition. Compared with SCZ c/SM+, SCZ c/SM- patients were characterized by significantly more severe PANSS total, positive, disorganized/cognitive and excited psychopathology. Cognitive performance was also significantly poorer in this subgroup. These data show that a specific RBC membrane lipid cluster is associated with clinical and cognitive manifestations of dopamine dysfunction in schizophrenia patients. We speculate that this membrane lipid abnormality influences presynaptic dopamine signaling.

  19. A layer model of ethanol partitioning into lipid membranes.

    PubMed

    Nizza, David T; Gawrisch, Klaus

    2009-06-01

    The effect of membrane composition on ethanol partitioning into lipid bilayers was assessed by headspace gas chromatography. A series of model membranes with different compositions have been investigated. Membranes were exposed to a physiological ethanol concentration of 20 mmol/l. The concentration of membranes was 20 wt% which roughly corresponds to values found in tissue. Partitioning depended on the chemical nature of polar groups at the lipid/water interface. Compared to phosphatidylcholine, lipids with headgroups containing phosphatidylglycerol, phosphatidylserine, and sphingomyelin showed enhanced partitioning while headgroups containing phosphatidylethanolamine resulted in a lower partition coefficient. The molar partition coefficient was independent of a membrane's hydrophobic volume. This observation is in agreement with our previously published NMR results which showed that ethanol resides almost exclusively within the membrane/water interface. At an ethanol concentration of 20 mmol/l in water, ethanol concentrations at the lipid/water interface are in the range from 30-15 mmol/l, corresponding to one ethanol molecule per 100-200 lipids.

  20. Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

    PubMed Central

    Richards, Donald E.; Irvine, Robin F.; Dawson, Rex M. C.

    1979-01-01

    (1) The hydrolysis of 32P- or myo-[2-3H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca2+ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca2+ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[3H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed. PMID:508301

  1. Dietary lipids differentially affect membranes from different areas of rooster sperm.

    PubMed

    Bongalhardo, D C; Leeson, S; Buhr, M M

    2009-05-01

    The present work aimed to compare the effect of dietary flax with other oil sources on rooster sperm membranes and on semen characteristics. White Leghorn roosters (16 per diet) were fed 1 of 4 treatments: control diet (CON), or a diet containing corn oil (CORN), fish oil (FISH), or flax seed (FLAX) as the lipid source. Semen from 4 birds (30 wk old) of each treatment was pooled, the sperm head (HM) and body membranes (BM) were isolated, and lipids were extracted and analyzed. Aspects of lipid composition tested were as follows: percentage of individual fatty acids (C14:0 to C24:1) in total fatty acids, percentage of fatty acid categories [saturated, monounsaturated, polyunsaturated (PUFA), n-3 and n-6 PUFA, and n-6:n-3 ratio] within total fatty acids, and percentage of phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidylserine, and sphingomyelin] in total phospholipids. Sperm characteristics evaluated were as follows: volume, concentration, viability, percentage of motile cells, average path velocity, track speed, progressive velocity, lateral head displacement, straightness, and linearity. Diet did not affect membrane phospholipid ratios in either membrane but modified major fatty acids within certain phospholipids. Birds fed FISH and CORN showed, respectively, the highest and the lowest n-3 in sperm, causing reciprocal significant changes in n-6:n-3 ratio. Feeding FLAX caused intermediate effects in n-3, with values significantly lower than FISH but higher than CORN in HM (PC, PE, and phosphatidylinositol) and PC in BM (P < 0.05). In the PE phospholipids, FISH, followed by FLAX, increased n-3 in BM and decreased n-6 PUFA in HM. Sperm concentration was specifically correlated with the amount of 20:4n-6 in FLAX and 22:4n-6 in CON. In FLAX diets, straightness correlated with C18:0, n-3, and n-6:n-3 ratio. Diets containing distinct lipid sources differentially modify the lipid contents of HM and BM, with minor

  2. A Layer Model of Ethanol Partitioning into Lipid Membranes

    PubMed Central

    Nizza, David T.; Gawrisch, Klaus

    2013-01-01

    The effect of membrane composition on ethanol partitioning into lipid bilayers was assessed by headspace gas chromatography. A series of model membranes with different compositions have been investigated. Membranes were exposed to a physiological ethanol concentration of 20 mmol/l. The concentration of membranes was 20 wt% which roughly corresponds to values found in tissue. Partitioning depended on the chemical nature of polar groups at the lipid-water interface. Compared to phosphatidylcholine, lipids with headgroups containing phosphatidylglycerol, phosphatidylserine, and sphingomyelin showed enhanced partitioning while headgroups containing phosphatidylethanolamine resulted in a lower partition coefficient. The molar partition coefficient was independent of a membrane’s hydrophobic volume. This observation is in agreement with our previously published NMR results which showed that ethanol resides almost exclusively within the membrane-water interface. At an ethanol concentration of 20 mmol/l in water, ethanol concentrations at the lipid/water interface are in the range from 30 – 15 mmol/l, corresponding to one ethanol molecule per 100–200 lipids. PMID:19592710

  3. Complex Effects of 24:1 Sphingolipids in Membranes Containing Dioleoylphosphatidylcholine and Cholesterol.

    PubMed

    García-Arribas, Aritz B; González-Ramírez, Emilio J; Sot, Jesús; Areso, Itziar; Alonso, Alicia; Goñi, Félix M

    2017-06-06

    The effects of C24:1 sphingolipids have been tested in phospholipid bilayers containing cholesterol. Confocal microscopy, differential scanning calorimetry, and atomic force microscopy imaging and force curves have been used. More precisely, the effects of C24:1 ceramide (nervonoyl ceramide, nCer) were evaluated and compared to those of C16:0 ceramide (palmitoyl ceramide, pCer) in bilayers composed basically of dioleoylphosphatidylcholine, sphingomyelin (either C24:1, nSM or C16:0, pSM) and cholesterol. Combination of equimolecular amounts of C24:1 and C16:0 sphingolipids were also studied under the same conditions. Results show that both pCer and nCer are capable of forming segregated gel domains. Force spectroscopy data point to nCer having a lower stiffening effect than pCer, while the presence of nSM reduces the stiffness. DSC reveals T m reduction by nSM in every case. Furthermore, pSM seems to better accommodate both ceramides in a single phase of intermediate properties, while nSM partial accommodation of ceramides generates different gel phases with higher stiffnesses caused by interceramide cooperation. If both pSM and nSM are present, a clear preference of both ceramides toward pSM is observed. These findings show the sharp increase in complexity when membranes exhibit different sphingolipids of varying N-acyl chains, which should be a common issue in an actual cell membrane environment.

  4. Thyrotropin receptor and membrane interactions in FRTL-5 thyroid cell strain in microgravity.

    PubMed

    Albi, E; Ambesi-Impiombato, F S; Peverini, M; Damaskopoulou, E; Fontanini, E; Lazzarini, R; Curcio, F; Perrella, G

    2011-01-01

    The aim of this work was to analyze the possible alteration of thyrotropin (TSH) receptors in microgravity, which could explain the absence of thyroid cell proliferation in the space environment. Several forms of the TSH receptor are localized on the plasma membrane associated with caveolae and lipid rafts. The TSH regulates the fluidity of the cell membrane and the presence of its receptors in microdomains that are rich in sphingomyelin and cholesterol. TSH also stimulates cyclic adenosine monophosphate (cAMP) accumulation and cell proliferation. Reported here are the results of an experiment in which the FRTL-5 thyroid cell line was exposed to microgravity during the Texus-44 mission (launched February 7, 2008, from Kiruna, Sweden). When the parabolic flight brought the sounding rocket to an altitude of 264 km, the culture media were injected with or without TSH in the different samples, and weightlessness prevailed on board for 6 minutes and 19 seconds. Control experiments were performed, in parallel, in an onboard 1g centrifuge and on the ground in Kiruna laboratory. Cell morphology and function were analyzed. Results show that in microgravity conditions the cells do not respond to TSH treatment and present an irregular shape with condensed chromatin, a modification of the cell membrane with shedding of the TSH receptor in the culture medium, and an increase of sphingomyelin-synthase and Bax proteins. It is possible that real microgravity induces a rearrangement of specific sections of the cell membrane, which act as platforms for molecular receptors, thus influencing thyroid cell function in astronauts during space missions.

  5. Thyrotropin Receptor and Membrane Interactions in FRTL-5 Thyroid Cell Strain in Microgravity

    NASA Astrophysics Data System (ADS)

    Albi, E.; Ambesi-Impiombato, F. S.; Peverini, M.; Damaskopoulou, E.; Fontanini, E.; Lazzarini, R.; Curcio, F.; Perrella, G.

    2011-01-01

    The aim of this work was to analyze the possible alteration of thyrotropin (TSH) receptors in microgravity, which could explain the absence of thyroid cell proliferation in the space environment. Several forms of the TSH receptor are localized on the plasma membrane associated with caveolae and lipid rafts. The TSH regulates the fluidity of the cell membrane and the presence of its receptors in microdomains that are rich in sphingomyelin and cholesterol. TSH also stimulates cyclic adenosine monophosphate (cAMP) accumulation and cell proliferation. Reported here are the results of an experiment in which the FRTL-5 thyroid cell line was exposed to microgravity during the Texus-44 mission (launched February 7, 2008, from Kiruna, Sweden). When the parabolic flight brought the sounding rocket to an altitude of 264km, the culture media were injected with or without TSH in the different samples, and weightlessness prevailed on board for 6 minutes and 19 seconds. Control experiments were performed, in parallel, in an onboard 1g centrifuge and on the ground in Kiruna laboratory. Cell morphology and function were analyzed. Results show that in microgravity conditions the cells do not respond to TSH treatment and present an irregular shape with condensed chromatin, a modification of the cell membrane with shedding of the TSH receptor in the culture medium, and an increase of sphingomyelin-synthase and Bax proteins. It is possible that real microgravity induces a rearrangement of specific sections of the cell membrane, which act as platforms for molecular receptors, thus influencing thyroid cell function in astronauts during space missions.

  6. Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes-The Update.

    PubMed

    Maekawa, Masashi

    2017-03-03

    The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

  7. Co-evolution of sphingomyelin and the ceramide transport protein CERT.

    PubMed

    Hanada, Kentaro

    2014-05-01

    Life creates many varieties of lipids. The choline-containing sphingophospholipid sphingomyelin (SM) exists ubiquitously or widely in vertebrates and lower animals, but is absent or rare in bacteria, fungi, protists, and plants. In the biosynthesis of SM, ceramide, which is synthesized in the endoplasmic reticulum, is transported to the Golgi region by the ceramide transport protein CERT, probably in a non-vesicular manner, and is then converted to SM by SM synthase, which catalyzes the reaction of phosphocholine transfer from phosphatidylcholine (PtdCho) to ceramide. Recent advances in genomics and lipidomics indicate that the phylogenetic occurrence of CERT and its orthologs is nearly parallel to that of SM. Based on the chemistry of lipids together with evolutionary aspects of SM and CERT, several concepts are here proposed. SM may serve as a chemically inert and robust, but non-covalently interactive lipid class at the outer leaflet of the plasma membrane. The functional domains and peptidic motifs of CERT are separated by exon units, suggesting an exon-shuffling mechanism for the generation of an ancestral CERT gene. CERT may have co-evolved with SM to bypass a competing metabolic reaction at the bifurcated point in the anabolism of ceramide. Human CERT is identical to the splicing variant of human Goodpasture antigen-binding protein (GPBP) annotated as an extracellular non-canonical serine/threonine protein kinase. The relationship between CERT and GPBP has also been discussed from an evolutionary aspect. Moreover, using an analogy of "compatible (or osmoprotective) solutes" that can accumulate to very high concentrations in the cytosol without denaturing proteins, choline phospholipids such as PtdCho and SM may act as compatible phospholipids in biomembranes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology. © 2013.

  8. COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

    PubMed Central

    Meldolesi, J.; Jamieson, J. D.; Palade, G. E.

    1971-01-01

    The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ∼20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant. PMID:5555573

  9. Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

    PubMed

    Usery, Rebecca D; Enoki, Thais A; Wickramasinghe, Sanjula P; Nguyen, V P; Ackerman, David G; Greathouse, Denise V; Koeppe, Roger E; Barrera, Francisco N; Feigenson, Gerald W

    2018-05-08

    A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

    NASA Astrophysics Data System (ADS)

    Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R.; Oker-Blom, Christian; Vattulainen, Ilpo; Vuento, Matti

    2009-12-01

    In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter SCD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane.

  11. Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L.).

    PubMed

    Brogden, Graham; Propsting, Marcus; Adamek, Mikolaj; Naim, Hassan Y; Steinhagen, Dieter

    2014-03-01

    Cell membranes act as an interface between the interior of the cell and the exterior environment and facilitate a range of essential functions including cell signalling, cell structure, nutrient uptake and protection. It is composed of a lipid bilayer with integrated proteins, and the inner leaflet of the lipid bilayer comprises of liquid ordered (Lo) and liquid disordered (Ld) domains. Lo microdomains, also named as lipid rafts are enriched in cholesterol, sphingomyelin and certain types of proteins, which facilitate cell signalling and nutrient uptake. Lipid rafts have been extensively researched in mammals and the presence of functional lipid rafts was recently demonstrated in goldfish, but there is currently very little knowledge about their composition and function in fish. Therefore a protocol was established for the analysis of lipid rafts and membranous lipids in common carp (Cyprinus carpio L.) tissues. Twelve lipids were identified and analysed in the Ld domain of the membrane with the most predominant lipids found in all tissues being; triglycerides, cholesterol, phosphoethanolamine and phosphatidylcholine. Four lipids were identified in lipid rafts in all tissues analysed, triglycerides (33-62%) always found in the highest concentration followed by cholesterol (24-32%), phosphatidylcholine and sphingomyelin. Isolation of lipid rafts was confirmed by identifying the presence of the lipid raft associated protein flotillin, present at higher concentrations in the detergent resistant fraction. The data provided here build a lipid library of important carp tissues as a baseline for further studies into virus entry, protein trafficking or environmental stress analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses.

    PubMed

    Iwabuchi, Kazuhisa; Nakayama, Hitoshi; Masuda, Hiromi; Kina, Katsunari; Ogawa, Hideoki; Takamori, Kenji

    2012-01-01

    Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  13. N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

    PubMed

    Shaikh, Saame Raza; Teague, Heather

    2012-12-01

    This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

    PubMed

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-28

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  15. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

    NASA Astrophysics Data System (ADS)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-01

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  16. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

    PubMed Central

    Kim, JiHyun; Huang, Zhen; St. Clair, Johnna R.; Brown, Deborah A.; London, Erwin

    2016-01-01

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids. PMID:27872310

  17. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    PubMed

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  18. Orientation and Order of the Amide Group of Sphingomyelin in Bilayers Determined by Solid-State NMR

    PubMed Central

    Matsumori, Nobuaki; Yamaguchi, Toshiyuki; Maeta, Yoshiko; Murata, Michio

    2015-01-01

    Sphingomyelin (SM) and cholesterol (Chol) are considered essential for the formation of lipid rafts; however, the types of molecular interactions involved in this process, such as intermolecular hydrogen bonding, are not well understood. Since, unlike other phospholipids, SM is characterized by the presence of an amide group, it is essential to determine the orientation of the amide and its order in the lipid bilayers to understand the nature of the hydrogen bonds in lipid rafts. For this study, 1′-13C-2-15N-labeled and 2′-13C-2-15N-labeled SMs were prepared, and the rotational-axis direction and order parameters of the SM amide in bilayers were determined based on 13C and 15N chemical-shift anisotropies and intramolecular 13C-15N dipole coupling constants. Results revealed that the amide orientation was minimally affected by Chol, whereas the order was enhanced significantly in its presence. Thus, Chol likely promotes the formation of an intermolecular hydrogen-bond network involving the SM amide without significantly changing its orientation, providing a higher order to the SM amide. To our knowledge, this study offers new insight into the significance of the SM amide orientation with regard to molecular recognition in lipid rafts, and therefore provides a deeper understanding of the mechanism of their formation. PMID:26083921

  19. The metabolism of the phosphonium analogue of choline in cultured cells. A useful nuclear-magnetic-resonance probe for membrane phosphatidylcholine.

    PubMed Central

    Sim, E; Pasternak, C A

    1976-01-01

    1. Replacement of choline by the phosphonium analogue does not affect the growth rate of P815Y, NIL, 3T3, and SV40/3T3 cells in culture. 2. The fatty acid composition of the resulting phosphonium phosphatidylcholine is similar to that of phosphatidylcholine. 3. The rate of synthesis and degradation of phosphatidylcholine and of the phosphonium analogue are similar. 4. Phospholipid-exchange protein does not distinguish between phosphatidylcholine and the phosphonium analogue. 5. By contrast, incorporation of phosphonium choline into sphingomyelin occurs to only a minor extent. 6. It is concluded that, since the enzymes involved in the turnover of phosphatidylcholine do not discriminate between quaternary N and quaternary P in the polar head-group region, phosphonium choline should prove to be a useful probe for 31P nuclear-magnetic-resonance (n.m.r.) studies of natural membranes. PMID:1275902

  20. Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral.

    PubMed

    Chen, C-S; Lin, H-P; Yeh, C-C; Fang, L-S

    2005-12-01

    Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C(5)-DMB-SM). Incubation of symbiotic endoderm cells with C(5)-DMB-SM-defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research.

  1. Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

    PubMed

    Dinkla, S; Wessels, K; Verdurmen, W P R; Tomelleri, C; Cluitmans, J C A; Fransen, J; Fuchs, B; Schiller, J; Joosten, I; Brock, R; Bosman, G J C G M

    2012-10-18

    Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

  2. Sphingomyelin lipidosis (Niemann-Pick disease) in a juvenile raccoon (Procyon lotor).

    PubMed

    Vapniarsky, N; Wenger, D A; Scheenstra, D; Mete, A

    2013-01-01

    A wild caught juvenile male raccoon with neurological disease was humanely destroyed due to poor prognosis. Necropsy examination revealed hepatomegaly, splenomegaly and multicentric lymphadenomegaly with diffuse hepatic pallor and pulmonary consolidation with pinpoint pale subpleural foci. Microscopically, there was marked pale cytoplasmic swelling of the central and peripheral neurons as well as the glial cells in the brain, accompanied by multiorgan infiltration by abundant foamy macrophages. Ultrastructural investigation revealed accumulation of concentrically arranged lamellar material within lysosomes of the affected neurons, macrophages and endothelial cells. Biochemical enzymatic analysis detected sphingomyelinase deficiency and lysosomal storage disease consistent with sphingomyelin lipidosis (Niemann-Pick disease [NPD]) was diagnosed. This is the first report of NPD in a raccoon. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. [Evaluation of fetal lung maturity using a modified lecithin-sphingomyelin determination and Clements' foam test].

    PubMed

    Neumann, G; Gartzke, J; Faber, G

    1978-01-01

    The modified thin layer chromatographic method for the determination of the phospholipids lecithin and sphingomyelin from amniotic fluid is useful in estimating fetal pulmonary maturity. The foam test of Clements is a simple rapid method for screening of suspicious cases of pregnancies at risk and of great value as bed side test even performing by the doctor. In comparing Clements-Test with thin layer chromatographic for L/S-Ratio determination we found a good correlation of 81,8% of all cases.

  4. Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.

    PubMed

    Larsen, Jannik B; Kennard, Celeste; Pedersen, Søren L; Jensen, Knud J; Uline, Mark J; Hatzakis, Nikos S; Stamou, Dimitrios

    2017-09-19

    Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and

  5. Synthesis of asymmetric polyetherimide membrane for CO2/N2 separation

    NASA Astrophysics Data System (ADS)

    Ahmad, A. L.; Salaudeen, Y. O.; Jawad, Z. A.

    2017-06-01

    Large emission of carbon dioxide (CO2) to the environment requires mitigation to avoid unbearable consequences on global climate change. The CO2 emissions generated by fossil fuel combustion within the power and industrial sectors need to be quickly curbed. The gas emission can be abated using membrane technology; this is one of the most promising approaches for selective separation of CO2/N2. The purpose of the study is to synthesis an asymmetric polyetherimide (PEI) membrane and to establish its morphological characteristics for CO2/N2 separation. The PEI flat-sheet asymmetric membrane was fabricated using phase inversion with N-methyl-2-pyrrolidone (NMP) as solvent and water-isopropanol as a coagulant. Particularly, polymer concentration of 20, 25, and 30 wt. % were studied. In addition, the structure and morphology of the produced membrane were observed using scanning electron microscopy (SEM). Importantly, results showed that the membrane with high PEI concentration of 30 wt. % yield an optimal selectivity of 10.7 for CO2/Nitrogen (N2) separation at 1 bar and 25 ºC for pure gas, aided by the membrane surface morphology. The dense skin present was as a result of non-solvent (water) while isopropanol generates a porous sponge structure. This appreciable separation performance makes the PEI asymmetric membrane an attractive alternative for CO2/N2 separation.

  6. Regulation of sphingomyelin phosphodiesterase acid-like 3A gene (SMPDL3A) by liver X receptors.

    PubMed

    Noto, Paul B; Bukhtiyarov, Yuri; Shi, Meng; McKeever, Brian M; McGeehan, Gerard M; Lala, Deepak S

    2012-10-01

    Liver X receptor (LXR) α and LXRβ function as physiological sensors of cholesterol metabolites (oxysterols), regulating key genes involved in cholesterol and lipid metabolism. LXRs have been extensively studied in both human and rodent cell systems, revealing their potential therapeutic value in the contexts of atherosclerosis and inflammatory diseases. The LXR genome landscape has been investigated in murine macrophages but not in human THP-1 cells, which represent one of the frequently used monocyte/macrophage cell systems to study immune responses. We used a whole-genome screen to detect direct LXR target genes in THP-1 cells treated with two widely used LXR ligands [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317) and 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy] phenylacetic acid hydrochloride (GW3965)]. This screen identified the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene as a novel LXR-regulated gene, with an LXR response element within its promoter. We investigated the regulation of SMPDL3A gene expression by LXRs across several human and mouse cell types. These studies indicate that the induction of SMPDL3A is LXR-dependent and is restricted to human blood cells with no induction observed in mouse cellular systems.

  7. Digital holographic microscopy of phase separation in multicomponent lipid membranes

    NASA Astrophysics Data System (ADS)

    Farzam Rad, Vahideh; Moradi, Ali-Reza; Darudi, Ahmad; Tayebi, Lobat

    2016-12-01

    Lateral in-homogeneities in lipid compositions cause microdomains formation and change in the physical properties of biological membranes. With the presence of cholesterol and mixed species of lipids, phospholipid membranes segregate into lateral domains of liquid-ordered and liquid-disordered phases. Coupling of two-dimensional intralayer phase separations and interlayer liquid-crystalline ordering in multicomponent membranes has been previously demonstrated. By the use of digital holographic microscopy (DHMicroscopy), we quantitatively analyzed the volumetric dynamical behavior of such membranes. The specimens are lipid mixtures composed of sphingomyelin, cholesterol, and unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine. DHMicroscopy in a transmission mode is an effective tool for quantitative visualization of phase objects. By deriving the associated phase changes, three-dimensional information on the morphology variation of lipid stacks at arbitrary time scales is obtained. Moreover, the thickness distribution of the object at demanded axial planes can be obtained by numerical focusing. Our results show that the volume evolution of lipid domains follows approximately the same universal growth law of previously reported area evolution. However, the thickness of the domains does not alter significantly by time; therefore, the volume evolution is mostly attributed to the changes in area dynamics. These results might be useful in the field of membrane-based functional materials.

  8. Pharmacological Activation of Peroxisome Proliferator-Activated Receptor {Delta} Increases Sphingomyelin Synthase Activity in THP-1 Macrophage-Derived Foam Cell.

    PubMed

    Mou, Dongsheng; Yang, Hua; Qu, Changhua; Chen, Juan; Zhang, Chaogui

    2016-08-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which mediate glucose and lipid homeostasis by regulating the expression of a large number of transcription factors. Sphingomyelin synthase (SMS) is a key enzyme in the synthesis of sphingomyelin (SM), and its expression and activity have been reported to be associated with atherosclerosis (AS). Although there have been many functional PPAR and SMS studies on atherosclerosis in recent years, few have investigated the correlation between the activation of PPARδ and the activity of SMS. In his study, macrophage-induced foam cells were utilized to model important pathological changes that occur in AS. The influence of PPARδ agonism by GW501516 on SMS and its product molecule SM were measured. Results indicated that the activation of PPARδ was correlated in a positive manner with the activity of SMS2, and the content of SM was dose dependently increased by GW501516. Together, this study represents the first to suggest that PPARδ activation may be a potential risk of AS through enhancing activity of SMS2.

  9. The Chlamydial Inclusion Preferentially Intercepts Basolaterally Directed Sphingomyelin-Containing Exocytic Vacuoles

    PubMed Central

    Moore, Elizabeth R.; Fischer, Elizabeth R.; Mead, David J.; Hackstadt, Ted

    2010-01-01

    Chlamydiae replicate intracellularly within a unique vacuole termed the inclusion. The inclusion circumvents classical endosomal/lysosomal pathways but actively intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To further examine this interaction, we developed a polarized epithelial cell model to study vectoral trafficking of lipids and proteins to the inclusion. We examined seven epithelial cell lines for their ability to form single monolayers of polarized cells and support chlamydial development. Of these cell lines, polarized colonic mucosal C2BBe1 cells were readily infected with Chlamydia trachomatis and remained polarized throughout infection. Trafficking of (6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)hexanoyl)sphingosine) (NBD-C6-ceramide) and its metabolic derivatives, NBD-glucosylceramide (GlcCer) and NBD-SM, was analyzed. SM was retained within L2-infected cells relative to mock-infected cells, correlating with a disruption of basolateral SM trafficking. There was no net retention of GlcCer within L2-infected cells and purification of C. trachomatis elementary bodies from polarized C2BBe1 cells confirmed that bacteria retained only SM. The chlamydial inclusion thus appears to preferentially intercept basolaterally-directed SM-containing exocytic vesicles, suggesting a divergence in SM and GlcCer trafficking. The observed changes in lipid trafficking were a chlamydia-specific effect because Coxiella burnetii-infected cells revealed no changes in GlcCer or SM polarized trafficking. PMID:18778406

  10. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

    PubMed Central

    Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

    2006-01-01

    It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane. PMID:16858413

  11. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

    PubMed

    Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

    2006-07-26

    It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø <120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

  12. Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator.

    PubMed

    Miyamoto, Satoshi; Hsu, Cheng-Chih; Hamm, Gregory; Darshi, Manjula; Diamond-Stanic, Maggie; Declèves, Anne-Emilie; Slater, Larkin; Pennathur, Subramaniam; Stauber, Jonathan; Dorrestein, Pieter C; Sharma, Kumar

    2016-05-01

    AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Localization of cholesterol in sphingomyelinase-treated fibroblasts.

    PubMed Central

    Pörn, M I; Slotte, J P

    1995-01-01

    The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible

  14. Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms

    PubMed Central

    1994-01-01

    We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]- glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB- galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB

  15. Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy.

    PubMed

    Naito, Akira; Matsumori, Nobuaki; Ramamoorthy, Ayyalusamy

    2018-02-01

    A variety of biomolecules acting on the cell membrane folds into a biologically active structure in the membrane environment. It is, therefore, important to determine the structures and dynamics of such biomolecules in a membrane environment. While several biophysical techniques are used to obtain low-resolution information, solid-state NMR spectroscopy is one of the most powerful means for determining the structure and dynamics of membrane bound biomolecules such as antibacterial biomolecules and amyloidogenic proteins; unlike X-ray crystallography and solution NMR spectroscopy, applications of solid-state NMR spectroscopy are not limited by non-crystalline, non-soluble nature or molecular size of membrane-associated biomolecules. This review article focuses on the applications of solid-state NMR techniques to study a few selected antibacterial and amyloid peptides. Solid-state NMR studies revealing the membrane inserted bent α-helical structure associated with the hemolytic activity of bee venom melittin and the chemical shift oscillation analysis used to determine the transmembrane structure (with α-helix and 3 10 -helix in the N- and C-termini, respectively) of antibiotic peptide alamethicin are discussed in detail. Oligomerization of an amyloidogenic islet amyloid polypeptide (IAPP, or also known as amylin) resulting from its aggregation in a membrane environment, molecular interactions of the antifungal natural product amphotericin B with ergosterol in lipid bilayers, and the mechanism of lipid raft formation by sphingomyelin studied using solid state NMR methods are also discussed in this review article. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Biophysical implications of sphingosine accumulation in membrane properties at neutral and acidic pH.

    PubMed

    Zupancic, Eva; Carreira, Ana C; de Almeida, Rodrigo F M; Silva, Liana C

    2014-05-08

    Sphingosine (Sph) is a simple lipid involved in the regulation of several biological processes. When accumulated in the late endosomal/lysosomal compartments, Sph causes changes in ion signaling and membrane trafficking, leading to the development of Niemann-Pick disease type C. Little is known about Sph interaction with other lipids in biological membranes; however, understanding the effect of Sph in the physical state of membranes might provide insights into its mode of action. Using complementary established fluorescence approaches, we show that Sph accumulation leads to the formation of Sph-enriched gel domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (Chol) model membranes. These domains are more easily formed in membrane models mimicking the neutral pH plasma membrane environment (PM) as compared to the acidic lysosomal membrane environment (LM), where higher Sph concentrations (or lower temperatures) are required. Electrophoretic light scattering measurements further revealed that in PM-raft models (POPC/SM/Chol), Sph is mainly neutral, whereas in LM models, the positive charge of Sph leads to electrostatic repulsion, reducing the Sph ability to form gel domains. Thus, formation of Sph-enriched domains in cellular membranes might be strongly regulated by Sph charge.

  17. Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

    PubMed

    Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

    2018-01-01

    It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

  18. Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

    NASA Astrophysics Data System (ADS)

    Machta, Benjamin B.; Gray, Ellyn; Nouri, Mariam; McCarthy, Nicola L. C.; Gray, Erin M.; Miller, Ann L.; Brooks, Nicholas J.; Veatch, Sarah L.

    2016-08-01

    Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature ($T_c$) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises $T_c$ in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that $\\Delta T_c$ predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

  19. Highly Permeable AlPO-18 Membranes for N 2 /CH 4 Separation

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

    Zong, Zhaowang; Elsaidi, Sameh K.; Thallapally, Praveen K.

    Herein we demonstrate that AlPO-18 membranes can separate N2/CH4 gas mixtures at unprecedented N2 permeances. The best membranes separated N2/CH4 mixtures with N2 permeances as high as 3076 GPU and separation selectivities as high as 4.6. Gas mixture separation data, N2 and CH4 adsorption isotherms, ideal adsorbed solution theory (IAST), and breakthrough experiments were collected to understand the separation mechanisms. Competitive adsorption and differences in diffusivities were identified as the prevailing separation mechanisms. Differences in diffusivity played a more dominant role than the competitive adsorption, and led to nitrogen selective membranes.

  20. [Peculiarities of the phospholipid and fatty acid composition of erythrocyte plasma membranes of the Black Sea fish].

    PubMed

    Silkin, Iu A; Silkina, E N; Zabelinskiĭ, S A

    2012-01-01

    The phospholipid and the fatty acid composition of the main phospholipids families of erythrocyte plasma membranes was studied in two species of cartilaginous fish: the common thrasher (Raja clavata L.) and the common stingray (Dasyatis pastinaca) and three bony fish species: the scorpion fish (Scorpaena porcus L.), the smarida (Spicara flexuosa Raf.), and the horse mackerel (Trachurus mediterraneus ponticus Aleev). It was shown that in the studied fish, 70.0-80.0 % of all membrane phospholipids were composed of phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine, monophosphoinositide, and sphingomyelin were minor components whose content in the erythrocyte membrane fluctuated from 3.0 % to 13.0 %. The fatty acid phospholipids composition was represented by a large specter of acids. From saturated acids, basic for plasma membranes are palmitic (C16: 0) and stearic (C18: 0) acids. From unsaturated acids, the larger part belong to mono-, tetra-, penta-, and hexaenoic acids in fish phospholipids. The calculation of the double bond index and of the unsaturation coefficient showed difference in the deformation ability of erythrocyte membranes of the studied fish.

  1. Effect of long-term dietary sphingomyelin supplementation on atherosclerosis in mice

    PubMed Central

    Chung, Rosanna W. S.; Wang, Zeneng; Bursill, Christina A.; Wu, Ben J.; Barter, Philip J.

    2017-01-01

    Sphingomyelin (SM) levels in the circulation correlate positively with atherosclerosis burden. SM is a ubiquitous component of human diets, but it is unclear if dietary SM increases circulating SM levels. Dietary choline increases atherosclerosis by raising circulating trimethylamine N-oxide (TMAO) levels in mice and humans. As SM has a choline head group, we ask in this study if dietary SM accelerates atherosclerotic lesion development by increasing circulating SM and TMAO levels. Three studies were performed: (Study 1) C57BL/6 mice were maintained on a high fat diet with or without SM supplementation for 4 weeks prior to quantification of serum TMAO and SM levels; (Study 2) atherosclerosis was studied in apoE-/- mice after 16 weeks of a high fat diet without or with SM supplementation and (Study 3) apoE-/- mice were maintained on a chow diet for 19 weeks without or with SM supplementation and antibiotic treatment prior to quantification of atherosclerotic lesions and serum TMAO and SM levels. SM consumption did not increase circulating SM levels or atherosclerosis in high fat-fed apoE-/- mice. Serum TMAO levels in C57BL/6 mice were low and had no effect atherosclerosis lesion development. Dietary SM supplementation significantly reduced atherosclerotic lesion area in the aortic arch of chow-fed apoE-/- mice. This study establishes that dietary SM does not affect circulating SM levels or increase atherosclerosis in high fat-fed apoE-/- mice, but it is anti-atherogenic in chow-fed apoE-/- mice. PMID:29240800

  2. Visualization of Detergent Solubilization of Membranes: Implications for the Isolation of Rafts

    PubMed Central

    Garner, Ashley E.; Smith, D. Alastair; Hooper, Nigel M.

    2008-01-01

    Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (ld) and raft liquid ordered (lo) lipid phases by selectively solubilizing the ld phase. A higher concentration of Lubrol was required, and not all the ld phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some ld phase and then progressed to the solubilization of both ld and lo phases simultaneously. Octyl glucoside simultaneously solubilized both lo and ld phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems. PMID:17933878

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

    PubMed

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

    2016-01-11

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

  4. Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux.

    PubMed

    Leventhal, A R; Chen, W; Tall, A R; Tabas, I

    2001-11-30

    Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel

  5. 15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

    PubMed

    Lanquar, Viviane; Kuhn, Lauriane; Lelièvre, Françoise; Khafif, Mehdi; Espagne, Christelle; Bruley, Christophe; Barbier-Brygoo, Hélène; Garin, Jérôme; Thomine, Sébastien

    2007-03-01

    An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

  6. Biochemical characterization of detergent-resistant membranes: a systematic approach

    PubMed Central

    Babiychuk, Eduard B.; Draeger, Annette

    2006-01-01

    Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be ‘investigator-specific’. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the ‘brick-like’ raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data. PMID:16608442

  7. Biochemical characterization of detergent-resistant membranes: a systematic approach.

    PubMed

    Babiychuk, Eduard B; Draeger, Annette

    2006-08-01

    Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

  8. Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

    PubMed

    Nguyen, Khoa; Garcia, Alvaro; Sani, Marc-Antoine; Diaz, Dil; Dubey, Vikas; Clayton, Daniel; Dal Poggetto, Giovanni; Cornelius, Flemming; Payne, Richard J; Separovic, Frances; Khandelia, Himanshu; Clarke, Ronald J

    2018-06-01

    The Na + ,K + -ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na + and K + electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31 P and 2 H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Sphingomyelin in High-Density Lipoproteins: Structural Role and Biological Function

    PubMed Central

    Martínez-Beamonte, Roberto; Lou-Bonafonte, Jose M.; Martínez-Gracia, María V.; Osada, Jesús

    2013-01-01

    High-density lipoprotein (HDL) levels are an inverse risk factor for cardiovascular diseases, and sphingomyelin (SM) is the second most abundant phospholipid component and the major sphingolipid in HDL. Considering the marked presence of SM, the present review has focused on the current knowledge about this phospholipid by addressing its variable distribution among HDL lipoparticles, how they acquire this phospholipid, and the important role that SM plays in regulating their fluidity and cholesterol efflux from different cells. In addition, plasma enzymes involved in HDL metabolism such as lecithin–cholesterol acyltransferase or phospholipid transfer protein are inhibited by HDL SM content. Likewise, HDL SM levels are influenced by dietary maneuvers (source of protein or fat), drugs (statins or diuretics) and modified in diseases such as diabetes, renal failure or Niemann–Pick disease. Furthermore, increased levels of HDL SM have been shown to be an inverse risk factor for coronary heart disease. The complexity of SM species, described using new lipidomic methodologies, and their distribution in different HDL particles under many experimental conditions are promising avenues for further research in the future. PMID:23571495

  10. The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet.

    PubMed

    Tannert, Astrid; Kurz, Anke; Erlemann, Karl-Rudolf; Müller, Karin; Herrmann, Andreas; Schiller, Jürgen; Töpfer-Petersen, Edda; Manjunath, Puttaswamy; Müller, Peter

    2007-04-01

    The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109 to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species. Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented.

  11. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    PubMed

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

  12. ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

    PubMed Central

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters. PMID:25302608

  13. Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion

    PubMed Central

    Fernandes, Maria Cecilia; Cortez, Mauro; Flannery, Andrew R.; Tam, Christina; Mortara, Renato A.

    2011-01-01

    Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca2+ transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear. We show that host cell entry by T. cruzi mimics a process of plasma membrane injury and repair that involves Ca2+-dependent exocytosis of lysosomes, delivery of acid sphingomyelinase (ASM) to the outer leaflet of the plasma membrane, and a rapid form of endocytosis that internalizes membrane lesions. Host cells incubated with T. cruzi trypomastigotes are transiently wounded, show increased levels of endocytosis, and become more susceptible to infection when injured with pore-forming toxins. Inhibition or depletion of lysosomal ASM, which blocks plasma membrane repair, markedly reduces the susceptibility of host cells to T. cruzi invasion. Notably, extracellular addition of sphingomyelinase stimulates host cell endocytosis, enhances T. cruzi invasion, and restores normal invasion levels in ASM-depleted cells. Ceramide, the product of sphingomyelin hydrolysis, is detected in newly formed parasitophorous vacuoles containing trypomastigotes but not in the few parasite-containing vacuoles formed in ASM-depleted cells. Thus, T. cruzi subverts the ASM-dependent ceramide-enriched endosomes that function in plasma membrane repair to infect host cells. PMID:21536739

  14. Pathological levels of glucosylceramide change the biophysical properties of artificial and cell membranes.

    PubMed

    Varela, Ana R P; Ventura, Ana E; Carreira, Ana C; Fedorov, Aleksander; Futerman, Anthony H; Prieto, Manuel; Silva, Liana C

    2016-12-21

    Glucosylceramide (GlcCer) plays an active role in the regulation of various cellular events. Moreover, GlcCer is also a key modulator of membrane biophysical properties, which might be linked to the mechanism of its biological action. In order to understand the biophysical implications of GlcCer on membranes of living cells, we first studied the effect of GlcCer on artificial membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). Using an array of biophysical methods, we demonstrate that at lower GlcCer/Chol ratios, GlcCer stabilizes SM/Chol-enriched liquid-ordered domains. However, upon decreasing the Chol content, GlcCer significantly increased membrane order through the formation of gel domains. Changes in pH disturbed the packing properties of GlcCer-containing membranes, leading to an increase in membrane fluidity and reduced membrane electronegativity. To address the biophysical impact of GlcCer in biological membranes, studies were performed in wild type and in fibroblasts treated with conduritol-B-epoxide (CBE), which causes intracellular GlcCer accumulation, and in fibroblasts from patients with type I Gaucher disease (GD). Decreased membrane fluidity was observed in cells containing higher levels of GlcCer, such as in CBE-treated and GD cells. Together, we demonstrate that elevated GlcCer levels change the biophysical properties of cellular membranes, which might compromise membrane-associated cellular events and be of relevance for understanding the pathology of diseases, such as GD, in which GlcCer accumulates at high levels.

  15. Effects of Vanadium-Containing Compounds on Membrane Lipids and on Microdomains Used in Receptor-Mediated Signaling

    PubMed Central

    Roess, Deborah A.; Smith, Steven M. L.; Winter, Peter; Zhou, Jun; Dou, Ping; Baruah, Bharat; Trujillo, Alejandro M.; Levinger, Nancy E.; Yang, Xioda; Barisas, B. George; Crans, Debbie C.

    2011-01-01

    There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of vanadium compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving vanadium complexes such as [VO2dipic]− (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V10O286−, V10), BMOV (bis(maltolato)oxovanadium(IV)) and [VO(saltris)]2 (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between vanadium-containing compounds and model lipid systems, an evaluation of the effects of vanadium compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of vanadium-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms. PMID:18729092

  16. Dietary n-6:n-3 ratio and Vitamin E improve motility characteristics in association with membrane properties of boar spermatozoa.

    PubMed

    Liu, Qing; Zhou, Yuan-Fei; Duan, Run-Jia; Wei, Hong-Kui; Peng, Jian; Jiang, Si-Wen

    2017-01-01

    This study was aimed to evaluate the effects of dietary n-6:n-3 ratio and Vitamin E on the membrane properties and motility characteristics of spermatozoa in boars. Forty Duroc boars were randomly distributed in a 2 × 2 factorial design with two n-6:n-3 ratios (14.4 and 6.6) and two Vitamin E levels (200 and 400 mg kg-1 ). During 16 weeks of treatment, fresh semen was collected at weeks 0, 8, 12, and 16 for measurements of motility characteristics, contents of fatty acids, membrane properties (membrane fluidity and membrane integrity), and lipid peroxidation of the spermatozoa. The semen was diluted in Beltsville Thawing Solution (BTS) extender and stored at 17°C, and the sperm motility was assessed at 12, 36, 72, and 120 h of storage. The 6.6 n-6:n-3 dietary ratio increased the contents of n-3 polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid (DHA) and improved the membrane integrity and membrane fluidity of the spermatozoa, resulting in notably increased total motility, sperm progressive motility, and velocity parameters of fresh semen. Feeding diet with Vitamin E (400 mg kg-1 ) prevented sperm lipid peroxidation, and resulted in higher total motility and sperm progressive motility in fresh and liquid stored semen. In conclusion, the adjustment of n-6:n-3 ratio (6.6) and supply of Vitamin E (400 mg kg-1 ) successfully improved sperm motility characteristics and thus may be beneficial to the fertility of boars, which might be due to the modification of the physical and functional properties of spermatozoa membrane in response to dietary supplementation.

  17. Dietary n-6:n-3 ratio and Vitamin E improve motility characteristics in association with membrane properties of boar spermatozoa

    PubMed Central

    Liu, Qing; Zhou, Yuan-Fei; Duan, Run-Jia; Wei, Hong-Kui; Peng, Jian; Jiang, Si-Wen

    2017-01-01

    This study was aimed to evaluate the effects of dietary n-6:n-3 ratio and Vitamin E on the membrane properties and motility characteristics of spermatozoa in boars. Forty Duroc boars were randomly distributed in a 2 × 2 factorial design with two n-6:n-3 ratios (14.4 and 6.6) and two Vitamin E levels (200 and 400 mg kg−1). During 16 weeks of treatment, fresh semen was collected at weeks 0, 8, 12, and 16 for measurements of motility characteristics, contents of fatty acids, membrane properties (membrane fluidity and membrane integrity), and lipid peroxidation of the spermatozoa. The semen was diluted in Beltsville Thawing Solution (BTS) extender and stored at 17°C, and the sperm motility was assessed at 12, 36, 72, and 120 h of storage. The 6.6 n-6:n-3 dietary ratio increased the contents of n-3 polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid (DHA) and improved the membrane integrity and membrane fluidity of the spermatozoa, resulting in notably increased total motility, sperm progressive motility, and velocity parameters of fresh semen. Feeding diet with Vitamin E (400 mg kg−1) prevented sperm lipid peroxidation, and resulted in higher total motility and sperm progressive motility in fresh and liquid stored semen. In conclusion, the adjustment of n-6:n-3 ratio (6.6) and supply of Vitamin E (400 mg kg−1) successfully improved sperm motility characteristics and thus may be beneficial to the fertility of boars, which might be due to the modification of the physical and functional properties of spermatozoa membrane in response to dietary supplementation. PMID:26763547

  18. α- tocopherol’s location in membranes is not affected by their composition

    DOE PAGES

    Marquardt, Drew; Kucerka, Norbert; Katsaras, John; ...

    2014-10-15

    To this day, α-tocopherol's (aToc) role in humans is not well known. In previous studies, we have tried to connect aToc's biological function with its location in a-lipid bilayer. In the present study, we have determined, by means of small-angle neutron diffraction, that not only is aToc's hydroxyl group located high in the membrane but its tail also resides far from the center of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. In addition, we located aToc's hydroxyl group above the lipid backbone in 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-L-serine (POPS), and Sphingomyelin bilayers, suggesting that aToc's location near the lipid water interface may be amore » universal property of vitamin E. Lastly, in light of these data, how aToc efficiently terminates lipid hydroperoxy radicals at the membrane center remains an open question.« less

  19. GaN membrane MSM ultraviolet photodetectors

    NASA Astrophysics Data System (ADS)

    Muller, A.; Konstantinidis, G.; Kostopoulos, A.; Dragoman, M.; Neculoiu, D.; Androulidaki, M.; Kayambaki, M.; Vasilache, D.; Buiculescu, C.; Petrini, I.

    2006-12-01

    GaN exhibits unique physical properties, which make this material very attractive for wide range of applications and among them ultraviolet detection. For the first time a MSM type UV photodetector structure was manufactured on a 2.2 μm. thick GaN membrane obtained using micromachining techniques. The low unintentionally doped GaN layer structure was grown by MOCVD on high resistivity (ρ>10kΩcm) <111> oriented silicon wafers, 500μm thick. The epitaxially grown layers include a thin AlN layer in order to reduce the stress in the GaN layer and avoid cracking. Conventional contact lithography, e-gun Ni/Au (10nm /200nm) evaporation and lift-off techniques were used to define the interdigitated Schottky metalization on the top of the wafer. Ten digits with a width of 1μm and a length of 100μm were defined for each electrode. The distance between the digits was also 1μm. After the backside lapping of the wafer to a thickness of approximately 150μm, a 400nm thick Al layer was patterned and deposited on the backside, to be used as mask for the selective reactive ion etching of silicon. The backside mask, for the membrane formation, was patterned using double side alignment techniques and silicon was etched down to the 2.2μm thin GaN layer using SF 6 plasma. A very low dark current (30ρA at 3V) was obtained. Optical responsivity measurements were performed at 1.5V. A maximum responsivity of 18mA/W was obtained at a wavelength of 370nm. This value is very good and can be further improved using transparent contacts for the interdigitated structure.

  20. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  1. Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes

    PubMed Central

    Štefl, Martin; Šachl, Radek; Humpolíčková, Jana; Cebecauer, Marek; Macháň, Radek; Kolářová, Marie; Johansson, Lennart B.-Å.; Hof, Martin

    2012-01-01

    Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ∼8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation. PMID:22824274

  2. One-step fabrication of porous GaN crystal membrane and its application in energy storage

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Wang, Shouzhi; Shao, Yongliang; Wu, Yongzhong; Sun, Changlong; Huo, Qin; Zhang, Baoguo; Hu, Haixiao; Hao, Xiaopeng

    2017-03-01

    Single-crystal gallium nitride (GaN) membranes have great potential for a variety of applications. However, fabrication of single-crystalline GaN membranes remains a challenge owing to its chemical inertness and mechanical hardness. This study prepares large-area, free-standing, and single-crystalline porous GaN membranes using a one-step high-temperature annealing technique for the first time. A promising separation model is proposed through a comprehensive study that combines thermodynamic theories analysis and experiments. Porous GaN crystal membrane is processed into supercapacitors, which exhibit stable cycling life, high-rate capability, and ultrahigh power density, to complete proof-of-concept demonstration of new energy storage application. Our results contribute to the study of GaN crystal membranes into a new stage related to the elelctrochemical energy storage application.

  3. Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure

    PubMed Central

    Dinkla, S; Wessels, K; Verdurmen, W P R; Tomelleri, C; Cluitmans, J C A; Fransen, J; Fuchs, B; Schiller, J; Joosten, I; Brock, R; Bosman, G J C G M

    2012-01-01

    Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane–cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane–cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions. PMID:23076218

  4. n-3 and n-6 Fatty Acid Changes in the Erythrocyte Membranes of Patients with 658240251 Clostridium difficile Infection.

    PubMed

    Czepiel, Jacek; Gdula-Argasińska, Joanna; Garlicki, Aleksander

    2016-01-01

    The implications of circulating essential fatty acids (FA) on the inflammatory risk profile and clinical outcome are still unclear. In order to gain a deeper understanding of the role of polyunsaturated fatty acids (PUFA) in the pathogenesis of acute infection, we analyzed the FA content in red blood cell (RBC) membranes of patients with Clostridium difficile infection (CDI) and controls. We prospectively studied 60 patients including 30 patients with CDI and 30 controls to assess lipid concentrations in erythrocyte membranes using gas chromatography. We observed a higher level of saturated fatty acids (SFA) in RBC membranes from patients with CDI. In patients with CDI, we also noticed a higher level of 20:4 n-6 FA and only a small amounts of C20:2n-6, C20:3n-6 FAs, arachidonic acid (AA) precursors, which suggest an intense inflammatory reaction in the organism during infection. We also noticed low levels of n-3 FA in the RBC membranes of patients infected with CDI. There is a deficit of n-3 FA in patients with CDI. n-3 FA are probably used during CDI as precursors of pro-resolving mediators that may indicate a therapeutic role of n-3 PUFAs in CDI. The changes in fatty acids in erythrocyte membranes during CDI alter their functions which may have an impact on the clinical outcome.

  5. Visualization of detergent solubilization of membranes: implications for the isolation of rafts.

    PubMed

    Garner, Ashley E; Smith, D Alastair; Hooper, Nigel M

    2008-02-15

    Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.

  6. Trans-membrane transport of n-octadecane by Pseudomonas sp. DG17.

    PubMed

    Hua, Fei; Wang, Hong Qi; Li, Yi; Zhao, Yi Cun

    2013-12-01

    The trans-membrane transport of hydrocarbons is an important and complex aspect of the process of biodegradation of hydrocarbons by microorganisms. The mechanism of transport of (14)C n-octadecane by Pseudomonas sp. DG17, an alkane-degrading bacterium, was studied by the addition of ATP inhibitors and different substrate concentrations. When the concentration of n-octadecane was higher than 4.54 μmol/L, the transport of (14)C n-octadecane was driven by a facilitated passive mechanism following the intra/extra substrate concentration gradient. However, when the cells were grown with a low concentration of the substrate, the cellular accumulation of n-octadecane, an energy-dependent process, was dramatically decreased by the presence of ATP inhibitors, and n-octadecane accumulation continually increased against its concentration gradient. Furthermore, the presence of non-labeled alkanes blocked (14)C n-octadecane transport only in the induced cells, and the trans-membrane transport of n-octadecane was specific with an apparent dissociation constant K t of 11.27 μmol/L and V max of 0.96 μmol/min/mg protein. The results indicated that the trans-membrane transport of n-octadecane by Pseudomonas sp. DG17 was related to the substrate concentration and ATP.

  7. Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

    PubMed

    Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

    2010-06-01

    The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

  8. Multi-step splicing of sphingomyelin synthase linear and circular RNAs.

    PubMed

    Filippenkov, Ivan B; Sudarkina, Olga Yu; Limborska, Svetlana A; Dergunova, Lyudmila V

    2018-05-15

    The SGMS1 gene encodes the enzyme sphingomyelin synthase 1 (SMS1), which is involved in the regulation of lipid metabolism, apoptosis, intracellular vesicular transport and other significant processes. The SGMS1 gene is located on chromosome 10 and has a size of 320 kb. Previously, we showed that dozens of alternative transcripts of the SGMS1 gene are present in various human tissues. In addition to mRNAs that provide synthesis of the SMS1 protein, this gene participates in the synthesis of non-coding transcripts, including circular RNAs (circRNAs), which include exons of the 5'-untranslated region (5'-UTR) and are highly represented in the brain. In this study, using the high-throughput technology RNA-CaptureSeq, many new SGMS1 transcripts were identified, including both intronic unspliced RNAs (premature RNAs) and RNAs formed via alternative splicing. Recursive exons (RS-exons) that can participate in the multi-step splicing of long introns of the gene were also identified. These exons participate in the formation of circRNAs. Thus, multi-step splicing may provide a variety of linear and circular RNAs of eukaryotic genes in tissues. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro

    PubMed Central

    Nusshold, Christoph; Kollroser, Manfred; Köfeler, Harald; Rechberger, Gerald; Reicher, Helga; Üllen, Andreas; Bernhart, Eva; Waltl, Sabine; Kratzer, Ingrid; Hermetter, Albin; Hackl, Hubert; Trajanoski, Zlatko; Hrzenjak, Andelko; Malle, Ernst; Sattler, Wolfgang

    2014-01-01

    Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H2O2 and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO–H2O2–chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis. PMID:20226853

  10. Interaction of miltefosine with the lipid and protein components of the erythrocyte membrane.

    PubMed

    Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Hansen, Daiane; Alonso, Antonio

    2013-05-01

    Miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although its mechanism of action remains poorly understood. Electron paramagnetic resonance spectroscopy of a spin-labeled lipid and a thiol-specific spin label showed that MT causes an increase in the molecular dynamics of erythrocyte ghost membranes and detergent-resistant membranes (DRMs) prepared from erythrocyte ghosts. In the vesicles of lipid raft constituents, it was shown that 20 mol % sphingomyelin could be replaced by 20 mol % MT with no change in the molecular dynamics. The effect of MT in DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-90 µg/mL), our measurements in the blood plasma indicated a hemolytic level of 2%-5%. The experiments indicated that MT acts predominantly on the protein component of the membrane. MT aggregates may wrap around the hydrophobic polypeptide chains, forming micelle-like structures that stabilize protein conformations more exposed to the solvent. Proteins with higher hydrophobicity may induce the penetration of the hydrophilic groups of MT into the membrane and cause it to rupture. Copyright © 2013 Wiley Periodicals, Inc.

  11. Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

    NASA Technical Reports Server (NTRS)

    Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1991-01-01

    Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

  12. The insertion and transport of anandamide in synthetic lipid membranes are both cholesterol-dependent.

    PubMed

    Di Pasquale, Eric; Chahinian, Henri; Sanchez, Patrick; Fantini, Jacques

    2009-01-01

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

  13. High rejection reverse osmosis membrane for removal of N-nitrosamines and their precursors.

    PubMed

    Fujioka, Takahiro; Ishida, Kenneth P; Shintani, Takuji; Kodamatani, Hitoshi

    2017-12-12

    Direct potable reuse is becoming a feasible option to cope with water shortages. It requires more stringent water quality assurance than indirect potable reuse. Thus, the development of a high-rejection reverse osmosis (RO) membrane for the removal of one of the most challenging chemicals in potable reuse - N-nitrosodimethylamine (NDMA) - ensures further system confidence in reclaimed water quality. This study aimed to achieve over 90% removal of NDMA by modifying three commercial and one prototype RO membrane using heat treatment. Application of heat treatment to a prototype membrane resulted in a record high removal of 92% (1.1-log) of NDMA. Heat treatment reduced conductivity rejection and permeability, while secondary amines, selected as N-nitrosamine precursors, were still well rejected (>98%) regardless of RO membrane type. This study also demonstrated the highly stable separation performance of the heat-treated prototype membrane under conditions of varying feed temperature and permeate flux. Fouling propensity of the prototype membrane was lower than a commercial RO membrane. This study identified a need to develop highly selective RO membranes with high permeability to ensure the feasibility of using these membranes at full scale. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

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

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The bindingmore » of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.« less

  15. Cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.

    PubMed

    Härmälä, A S; Pörn, M I; Mattjus, P; Slotte, J P

    1994-03-24

    The compound U1866A (3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one) has been shown to inhibit the cellular transfer of low-density lipoprotein-derived cholesterol from lysosomes to plasma membranes (Liscum and Faust (1989) J. Biol. Chem. 264, 11796-806). We have in this study examined the effects of U18666A on cholesterol translocation from plasma membranes to intracellular membranes. Translocation of plasma membrane cholesterol was induced by degradation of plasma membrane sphingomyelin. The sphingomyelinase-induced activation of the acyl-CoA cholesterol acyl transferase (ACAT) reaction was completely inhibited in a dose-dependent manner by U18666A, both in cultured human skin fibroblasts and baby hamster kidney cells. Half-maximal inhibition (within 60 min) was obtained with 0.5-1 microgram/ml of U18666A. A time-course study indicated that the onset of inhibition was rapid (within 10-15 min), and reversible if U18666A was removed from the incubation mixture. Using a cholesterol oxidase assay, we observed that the extent of plasma membrane cholesterol translocation in sphingomyelinase-treated HSF cells was significantly lowered in the presence of U18666A (at 3 micrograms/ml). The effect of U18666A on cholesterol translocation was also fully reversible when the drug was withdrawn. In mouse Leydig tumor cells, labeled to constant specific activity with [3H]cholesterol, the compound U18666A inhibited in a dose-dependent manner the cyclic AMP-stimulated secretion of [3H]steroid hormones. The effects seen with compound U18666A appeared to be specific for this molecule, since another hydrophobic amine, imipramine, did not in our experiments affect cholesterol translocation or ACAT activation. Since different cell types display sensitivity to U18666A in various intracellular cholesterol transfer processes, they appear to have a common U18666A-sensitive regulatory mechanism.

  16. Prediction of fetal lung maturity using the lecithin/sphingomyelin (L/S) ratio analysis with a simplified sample preparation, using a commercial microtip-column combined with mass spectrometric analysis.

    PubMed

    Kwak, Ho-Seok; Chung, Hee-Jung; Choi, Young Sik; Min, Won-Ki; Jung, So Young

    2015-07-01

    Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC-MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC-MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC-MS/MS (kappa value=1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value=1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC-MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles.

    PubMed

    Meyer, H W; Bunjes, H; Ulrich, A S

    1999-06-01

    The phase transition of hydrated brain sphingomyelin occurs at around 35 degrees C, which is close to the physiological temperature. Freeze-fracture electron microscopy is used to characterize different gel state morphologies in terms of solid-ordered and liquid-ordered phase states, according to the occurrence of ripples and other higher-dimensional bilayer deformations. Evidently, the natural mixed-chain sphingomyelin does not assume the flat L beta, phase but instead the rippled P beta, phase, with symmetric and asymmetric ripples as well as macroripples and an egg-carton pattern, depending on the incubation conditions. An unexpected difference was observed between samples that are hydrated above and below the phase transition temperature. When the lipid is hydrated at low temperature, a sponge-like network of bilayers is formed in the gel state, next to some normal lamellae. The network loses its ripples during cold-incubation, which indicates the formation of a liquid-ordered (lo) gel phase. Ripples re-appear upon warming and the sponge-like network disintegrates spontaneously and irreversibly into small vesicles above the phase transition.

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

    PubMed

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

    2013-08-06

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

  19. Preparation and photoelectrocatalytic performance of N-doped TiO2/NaY zeolite membrane composite electrode material.

    PubMed

    Cheng, Zhi-Lin; Han, Shuai

    2016-01-01

    A novel composite electrode material based on a N-doped TiO2-loaded NaY zeolite membrane (N-doped TiO2/NaY zeolite membrane) for photoelectrocatalysis was presented. X-ray diffraction (XRD), scanning electron microscopy (SEM), UV-visible (UV-vis) and X-ray photoelectron spectroscopy (XPS) characterization techniques were used to analyze the structure of the N-doped TiO2/NaY zeolite membrane. The XRD and SEM results verified that the N-doped TiO2 nanoparticles with the size of ca. 20 nm have been successfully loaded on the porous stainless steel-supported NaY zeolite membrane. The UV-vis result showed that the N-doped TiO2/NaY zeolite membrane exhibited a more obvious red-shift than that of N-TiO2 nanoparticles. The XPS characterization revealed that the doping of N element into TiO2 was successfully achieved. The photoelectrocatalysis performance of the N-doped TiO2/NaY zeolite membrane composite electrode material was evaluated by phenol removal and also the effects of reaction conditions on the catalytic performance were investigated. Owing to exhibiting an excellent catalytic activity and good recycling stability, the N-doped TiO2/NaY zeolite membrane composite electrode material was of promising application for photoelectrocatalysis in wastewater treatment.

  20. SAPO-34 Membranes for N-2/CH4 separation: Preparation, characterization, separation performance and economic evaluation

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

    Li, SG; Zong, ZW; Zhou, SJ

    2015-08-01

    SAPO-34 membranes were synthesized by several routes towards N-2/CH4 separation. Membrane synthesis parameters including water content in the gel, crystallization time, support pore size, and aluminum source were investigated. High performance N-2-selective membranes were obtained on 100-nm-pore alumina tubes by using Al(i-C3H7O)(3) as aluminum source with a crystallization time of 6 h. These membranes separated N-2 from CH, with N-2 permeance as high as 500 GPU with separation selectivity of 8 at 24 degrees C. for a 50/50 N-2/CH4 mixture. Nitrogen and CH, adsorption isotherms were measured on SAPO-34 crystals. The N-2 and CH, heats of adsorption were 11 andmore » 15 kJ/mol, respectively, which lead to a preferential adsorption of CE-H-4 over N-2 in the N-2/CH4 mixture. Despite this, the SAPO-34 membranes were selective for N-2 over CH4 in the mixture because N-2 diffuses much faster than CH4 and differences in diffusivity played a more critical role than the competitive adsorption. Preliminary economic evaluation indicates that the required N-2/CH4 selectivity would be 15 in order to maintain a CH4 loss below 10%. For small nitrogen-contaminated gas wells, our current SAPO-34 membranes have potential to compete with the benchmark technology cryogenic distillation for N-2 rejection. (C) 2015 Elsevier B.V. All rights reserved,« less

  1. Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins.

    PubMed

    García-Linares, Sara; Rivera-de-Torre, Esperanza; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M M; Gavilanes, José G; Slotte, J Peter; Martínez-Del-Pozo, Álvaro

    2016-12-06

    Sea anemone actinoporins constitute a protein family of multigene pore-forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three-dimensional structures. However, their pore-forming activity can be quite different depending on the model lipid system employed, a feature that has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore-forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed hemolytic activity much higher than those of the other three actinoporins studied. In lipid model systems, pore-forming ability when assayed against 4:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM) vesicles, with the membrane binding being the rate-limiting step, decreased in the following order: StnI > StnII > EqtII > FraC. When using 1:1:1 DOPC/SM/cholesterol LUVs, the presence of Chol not only enhanced membrane binding affinities by ∼2 orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N-terminal residues. The influence of interfacial hydrogen bonding in SM- or dihydro-SM-containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target

  2. Biophysical studies of cholesterol in unsaturated phospholipid model membranes

    NASA Astrophysics Data System (ADS)

    Williams, Justin Adam

    Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distributed to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. This research employs model membranes of well-defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega-3 (n-3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids. The molecular organization of 1-[2H31]palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC-d31) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31) in membranes with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid-state 2H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n-3 PUFAs found in fish oil, while PEPC-d31 and PDPC-d31 are phospholipids containing the respective PUFAs at the sn-2 position and a perdeuterated palmitic acid at the sn-1 position. Analysis of spectra recorded as a function of temperature indicates that in both cases, formation of PUFA-rich (less ordered) and SM-rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infiltrate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%). The implication is that n-3

  3. Role of membrane fouling substances on the rejection of N-nitrosamines by reverse osmosis.

    PubMed

    Fujioka, Takahiro; Kodamatani, Hitoshi; Aizawa, Hidenobu; Gray, Stephen; Ishida, Kenneth P; Nghiem, Long D

    2017-07-01

    The impact of fouling substances on the rejection of four N-nitrosamines by a reverse osmosis (RO) membrane was evaluated by characterizing individual organic fractions in a secondary wastewater effluent and deploying a novel high-performance liquid chromatography-photochemical reaction-chemiluminescence (HPLC-PR-CL) analytical technique. The HPLC-PR-CL analytical technique allowed for a systematic examination of the correlation between the fouling level and the permeation of N-nitrosamines in the secondary wastewater effluent and synthetic wastewaters through an RO membrane. Membrane fouling caused by the secondary wastewater effluent led to a notable decrease in the permeation of N-nitrosodimethylamine (NDMA) while a smaller but nevertheless discernible decrease in the permeation of N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPYR) and N-nitrosomorpholine (NMOR) was also observed. Fluorescence spectrometry analysis revealed that major foulants in the secondary wastewater effluent were humic and fulvic acid-like substances. Analysis using the size exclusion chromatography technique also identified polysaccharides and proteins as additional fouling substances. Thus, further examination was conducted using solutions containing model foulants (i.e., sodium alginate, bovine serum albumin, humic acid and two fulvic acids). Similar to the secondary wastewater effluent, membrane fouling with fulvic acid solutions resulted in a decrease in N-nitrosamine permeation. In contrast, membrane fouling with the other model foulants resulted in a negligible impact on N-nitrosamine permeation. Overall, these results suggest that the impact of fouling on the permeation of N-nitrosamines by RO is governed by specific small organic fractions (e.g. fulvic acid-like organics) in the secondary wastewater effluent. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Neuronal sphingolipidoses: Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism.

    PubMed

    Sandhoff, Konrad

    2016-11-01

    Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  5. Thermo-and pH-sensitive hydrogel membranes composed of poly(N-isopropylacrylamide)-hyaluronan for biomedical applications: Influence of hyaluronan incorporation on the membrane properties.

    PubMed

    Kamoun, Elbadawy A; Fahmy, Alaa; Taha, Tarek H; El-Fakharany, Esmail M; Makram, Mohamed; Soliman, Hesham M A; Shehata, Hassan

    2018-01-01

    Interpenetrating hydrogel membranes consisting of pH-sensitive hyaluronan (HA) and thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAM) were synthesized using redox polymerization, followed by N,N-methylenebisacrylamide (BIS) and epichlorohydrin (EPI) were added as chemical crosslinkers. The interaction between membrane compositions has been characterized by FTIR spectroscopy and discussed intensively. The result indicates that HA incorporation in membranes increase the gel fraction, swelling uptake, and the flexibility/elasticity of crosslinked membranes, however it reduced oppositely the mechanical elongation of membranes. PNIPAAm-HA hydrogels responded to both temperature and pH changes and the stimuli-responsiveness was reversible. However, in vitro bioevaluation results revealed that the released ampicillin during the burst release time was sharply influenced and increased with increasing HA contents in membranes; afterwards it became sustainable. Whereas, high HA contents in hydrogels unexpectedly impacted negatively on the cells viability, owing to the viscosity of cell culture media changed. A big resistance was observed against microbial growth of Staphylococcus aureus, Salmonella typhi, and Candida albicans in case of pure PNIPAAm hydrogel membranes without HA or ampicillin. However, HA incorporation or the loaded ampicillin in membranes showed unexpected easily microbial growth. The fast release performance with dual pH-thermo-sensitive hydrogels were suggested as promising materials for quick drug carrier in the biomedical field. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Ultrananocrystalline diamond-coated nanoporous membranes support SK-N-SH neuroblastoma endothelial cell attachment.

    PubMed

    Yang, Kai-Hung; Nguyen, Alexander K; Goering, Peter L; Sumant, Anirudha V; Narayan, Roger J

    2018-06-06

    Ultrananocrystalline diamond (UNCD) has been demonstrated to have attractive features for biomedical applications and can be combined with nanoporous membranes for applications in drug delivery systems, biosensing, immunoisolation and single molecule analysis. In this study, free-standing nanoporous UNCD membranes with pore sizes of 100 or 400 nm were fabricated by directly depositing ultrathin UNCD films on nanoporous silicon nitride membranes and then etching away silicon nitride using reactive ion etching. Successful deposition of UNCD on the substrate with a novel process was confirmed with Raman spectroscopy, X-ray photoelectron spectroscopy, cross-section scanning electron microscopy (SEM) and transmission electron microscopy. Both sample types exhibited uniform geometry and maintained a clear hexagonal pore arrangement. Cellular attachment of SK-N-SH neuroblastoma endothelial cells was examined using confocal microscopy and SEM. Attachment of SK-N-SH cells onto UNCD membranes on both porous regions and solid surfaces was shown, indicating the potential use of UNCD membranes in biomedical applications such as biosensors and tissue engineering scaffolds.

  7. Sea anemone actinoporins: the transition from a folded soluble state to a functionally active membrane-bound oligomeric pore.

    PubMed

    Alegre-Cebollada, J; Oñaderra, M; Gavilanes, J G; del Pozo, A Martínez

    2007-12-01

    Actinoporins are a family of 20-kDa, basic proteins isolated from sea anemones, whose activity is inhibited by preincubation with sphingomyelin. They are produced in monomeric soluble form but, when binding to the plasma membrane, they oligomerize to produce functional pores which result in cell lysis. Equinatoxin II (EqtII) from Actinia equina and Sticholysin II (StnII) from Stichodactyla helianthus are the actinoporins that have been studied in more detail. Both proteins display a beta-sandwich fold composed of 10 beta-strands flanked on each side by two short alpha-helices. Two-dimensional crystallization on lipid monolayers has allowed the determination of low-resolution models of tetrameric structures distinct from the pore. However, the actual structure of the pore is not known yet. Wild-type EqtII and StnII, as well as a nice collection of natural and artificially made variants of both proteins, have been produced in Escherichia coli and purified. Their characterization has allowed the proposal of a model for the mechanism of pore formation. Four regions of the actinoporins structure seem to play an important role. First, a phosphocholine-binding site and a cluster of exposed aromatic residues, together with a basic region, would be involved in the initial interaction with the membrane, whereas the amphipathic N-terminal region would be essential for oligomerization and pore formation. Accordingly, the model states that pore formation would proceed in at least four steps: Monomer binding to the membrane interface, assembly of four monomers, and at least two distinct conformational changes driving to the final formation of the functional pore.

  8. Macroscopic domain formation during cooling in the platelet plasma membrane: an issue of low cholesterol content

    PubMed Central

    Bali, Rachna; Savino, Laura; Ramirez, Diego A.; Tsvetkova, Nelly M.; Bagatolli, Luis; Tablin, Fern; Crowe, John H.; Leidy, Chad

    2009-01-01

    There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24°C, but not at 37°C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets (~15 mol %), appears to be crucial for the formation of large domains during cooling. PMID:19341703

  9. The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

    PubMed Central

    2009-01-01

    Background Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. Results Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. Conclusion The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm. PMID

  10. 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

  11. A biophysical approach to daunorubicin interaction with model membranes: relevance for the drug's biological activity.

    PubMed

    Alves, Ana Catarina; Ribeiro, Daniela; Horta, Miguel; Lima, José L F C; Nunes, Cláudia; Reis, Salette

    2017-08-01

    Daunorubicin is extensively used in chemotherapy for diverse types of cancer. Over the years, evidence has suggested that the mechanisms by which daunorubicin causes cytotoxic effects are also associated with interactions at the membrane level. The aim of the present work was to study the interplay between daunorubicin and mimetic membrane models composed of different ratios of 1,2-dimyristoyl- sn -glycero- 3 -phosphocholine (DMPC), sphingomyelin (SM) and cholesterol (Chol). Several biophysical parameters were assessed using liposomes as mimetic model membranes. Thereby, the ability of daunorubicin to partition into lipid bilayers, its apparent location within the membrane and its effect on membrane fluidity were investigated. The results showed that daunorubicin has higher affinity for lipid bilayers composed of DMPC, followed by DMPC : SM, DMPC : Chol and lastly by DMPC : SM : Chol. The addition of SM or Chol into DMPC membranes not only increases the complexity of the model membrane but also decreases its fluidity, which, in turn, reduces the amount of anticancer drug that can partition into these mimetic models. Fluorescence quenching studies suggest a broad distribution of the drug across the bilayer thickness, with a preferential location in the phospholipid tails. The gathered data support that daunorubicin permeates all types of membranes to different degrees, interacts with phospholipids through electrostatic and hydrophobic bonds and causes alterations in the biophysical properties of the bilayers, namely in membrane fluidity. In fact, a decrease in membrane fluidity can be observed in the acyl region of the phospholipids. Ultimately, such outcomes can be correlated with daunorubicin's biological action, where membrane structure and lipid composition have an important role. In fact, the results indicate that the intercalation of daunorubicin between the phospholipids can also take place in rigid domains, such as rafts that are known to be involved in

  12. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

    PubMed Central

    Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  13. Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

    PubMed

    Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

    2013-04-01

    Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013

  14. Carbamazepine degradation using a N-doped TiO2 coated photocatalytic membrane reactor: Influence of physical parameters.

    PubMed

    Horovitz, Inna; Avisar, Dror; Baker, Mark A; Grilli, Rossana; Lozzi, Luca; Di Camillo, Daniela; Mamane, Hadas

    2016-06-05

    Commercial α-Al2O3 photocatalytic membranes with a pore size of 200 and 800-nm were coated with N-doped TiO2 photocatalytic film using a sol-gel technique for concurrent bottom-up filtration and photocatalytic oxidation. X-ray diffraction confirmed that the deposited N-doped TiO2 films are in the form of anatase with 78-84% coverage of the membrane surface. The concentration of N found by X-ray photoelectron spectroscopy was in the range of 0.3-0.9 atomic percentage. Membrane permeability after coating decreased by 50% and 12% for the 200- and 800-nm membrane substrates, respectively. The impact of operational parameters on the photocatalytic activity (PCA) of the N-doped TiO2-coated membranes was examined in a laboratory flow cell based on degradation of the model micropollutant carbamazepine, using a solar simulator as the light source. The significant gap in degradation rate between flow through the membrane and flow on the surface of the membrane was attributed both to the hydraulic effect and in-pore PCA. N-doped TiO2-coated membranes showed enhanced activity for UV wavelengths, in addition to activity under visible light. Experiments of PCA under varying flow rates concluded that the process is in the mass-transfer control regime. Carbamazepine removal rate increased with temperature, despite the decrease in dissolved oxygen concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells.

    PubMed

    Regmi, Raju; Winkler, Pamina M; Flauraud, Valentin; Borgman, Kyra J E; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F

    2017-10-11

    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  16. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

    NASA Astrophysics Data System (ADS)

    Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

    2017-10-01

    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  17. Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

    PubMed

    Palestini, Paola; Calvi, Chiara; Conforti, Elena; Daffara, Rossella; Botto, Laura; Miserocchi, Giuseppe

    2003-10-01

    We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

  18. Membrane Alterations in Pseudomonas putida F1 Exposed to Nanoscale Zerovalent Iron: Effects of Short-Term and Repetitive nZVI Exposure.

    PubMed

    Kotchaplai, Panaya; Khan, Eakalak; Vangnai, Alisa S

    2017-07-18

    In this study, we report the effect of the commercial nanoscale zerovalent iron (nZVI) on environmental bacteria, emphasizing the importance of nZVI-bacterial membrane interaction on nZVI toxicity as well as the adaptability of bacteria to nZVI. Exposure of Pseudomonas putida F1 to 0.1, 1.0, and 5.0 g/L of nZVI caused the reduction in colony forming units (CFUs) substantially for almost 3 orders of magnitude. However, a rebound in the cell number was observed after the prolonged exposure except for 5.0 g/L nZVI at which bacterial viability was completely inhibited. Upon exposure, nZVI accumulated on and penetrated into the bacterial cell membrane. Cell membrane composition analysis revealed the conversion of the cis to trans isomer of unsaturated fatty acid upon short-term nZVI exposure, resulting in a more rigid membrane counteracting the membrane-fluidizing effect of nZVI. Several cycles of repetitive exposure of cells to 0.1 g/L nZVI induced a persistent phenotype of P. putida F1 as indicated by smaller colony morphology, a more rigid membrane, and higher tolerance to nZVI. A low interaction between nZVI particles and the surface of the nZVI-persistent phenotypic cells reduced the nZVI-induced membrane damage. This study unveils the significance of nZVI-membrane interaction on toxicity of nZVI toward bacteria.

  19. INF-gamma rearranges membrane topography of MHC-I and ICAM-1 in colon carcinoma cells.

    PubMed

    Bacsó, Zsolt; Bene, László; Damjanovich, László; Damjanovich, Sándor

    2002-01-18

    Flow-cytometric fluorescence energy transfer (FCET) measurements between fluorescently labeled cell surface MHC-I and ICAM-1 molecules indicated similar receptor patterns in the plasma membrane of interferon-gamma (INF-gamma)-treated colon carcinoma cells as those observed earlier at the surface of lymphoid cells. INF-gamma activation significantly increased the density of MHC-I and ICAM-1 proteins in the membrane. This increase in receptor density was accompanied by decreased proximity level of the homo-associated MHC-I receptors. Hetero-association of MHC-I and ICAM-1 molecules was increased by INF-gamma treatment. INF-gamma changed neither hetero- nor homo-association of transferrin receptors. By staining the sphingomyelin/cholesterol-enriched lipid microdomains with fluorescently labeled cholera toxin B subunit, we found an increase in the amount of lipid-raft associated G(M1)-gangliosides due to INF-gamma treatment. Confocal microscopic results and FCET measurements show that MHC-I and ICAM-1 are components of G(M1)-ganglioside containing lipid-rafts and also support an increase in the size of these lipid-rafts upon INF-gamma treatment.

  20. New Tethered Phospholipid Bilayers Integrating Functional G-Protein-Coupled Receptor Membrane Proteins.

    PubMed

    Chadli, Meriem; Rebaud, Samuel; Maniti, Ofelia; Tillier, Bruno; Cortès, Sandra; Girard-Egrot, Agnès

    2017-10-03

    Membrane proteins exhibiting extra- and intracellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19) possessing a cysteine residue in the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 2% molar ratio of a chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so-called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery after photobleaching (FRAP), with a diffusion coefficient of 2.5 × 10 -7 cm 2 /s, and atomic force microscopy (AFM). By using variable lipid compositions including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol 4,5-bisphosphate (PIP 2 ), sphingomyelin (SM), phosphatidic acid (PA), and cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing the functional C-X-C motif chemokine receptor 4 (CXCR4), a seven

  1. Study on CO2/ N2 separation: the effect of rubbery polymer coating on PVDF membrane

    NASA Astrophysics Data System (ADS)

    Zuwairi, M. Z.; Rahman, S. A.

    2017-06-01

    The emission of harmful gases such as carbon dioxide (CO2) via gas processing plant and daily human activities gave negative impacts to the environment and global inhabitant. Flat sheet asymmetric membranes were produced from homogenous solution of Poly(vinylideneflouride) (PVDF) via phase inversion method using N-methyl-2-pyrrolidone (NMP) as the solvent. While the poly ether b-amide (PEBAX) was dissolve by using of (70 ethanol and 30 water) as a solvent and and lithium chloride as a additives. The morphology and cross section of the produced membranes were observed by Scanning Electron Microscope (SEM). Then, the membranes were tested for chemical analysis to define the presence of PEBAX in the membrane by using Fourier Transform Infrared (FTIR) spectroscopy. The permeation performances of the membranes were evaluated in terms of permeability and selectivity of the membranes by using gas permeation test. Increasing the PEBAX content significantly increased the selectivity of the PVDF membrane to separate the CO2/N2 gases but decreased the amount of the gases that passed through the membrane.

  2. Al2O3 Disk Supported Si3N4 Hydrogen Purification Membrane for Low Temperature Polymer Electrolyte Membrane Fuel Cells.

    PubMed

    Liu, Xiaoteng; Christensen, Paul A; Kelly, Stephen M; Rocher, Vincent; Scott, Keith

    2013-12-05

    Reformate gas, a commonly employed fuel for polymer electrolyte membrane fuel cells (PEMFCs), contains carbon monoxide, which poisons Pt-containing anodes in such devices. A novel, low-cost mesoporous Si3N4 selective gas separation material was tested as a hydrogen clean-up membrane to remove CO from simulated feed gas to single-cell PEMFC, employing Nafion as the polymer electrolyte membrane. Polarization and power density measurements and gas chromatography showed a clear effect of separating the CO from the gas mixture; the performance and durability of the fuel cell was thereby significantly improved.

  3. Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect.

    PubMed

    Maddens, Stéphane; Charruyer, Alexandra; Plo, Isabelle; Dubreuil, Patrice; Berger, Stuart; Salles, Bernard; Laurent, Guy; Jaffrézou, Jean-Pierre

    2002-08-15

    Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.

  4. Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein

    PubMed Central

    2013-01-01

    Background Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production. Results To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism’s growth rate. Conclusions The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains. PMID:24020941

  5. Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein.

    PubMed

    Chin, Wei-Chih; Lin, Kuo-Hsing; Chang, Jui-Jen; Huang, Chieh-Chen

    2013-09-11

    Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production. To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism's growth rate. The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains.

  6. Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging*

    PubMed Central

    Subburaj, Yamunadevi; Ros, Uris; Hermann, Eduard; Tong, Rudi; García-Sáez, Ana J.

    2015-01-01

    α-Pore-forming toxins (α-PFTs) are ubiquitous defense tools that kill cells by opening pores in the target cell membrane. Despite their relevance in host/pathogen interactions, very little is known about the pore stoichiometry and assembly pathway leading to membrane permeabilization. Equinatoxin II (EqtII) is a model α-PFT from sea anemone that oligomerizes and forms pores in sphingomyelin-containing membranes. Here, we determined the spatiotemporal organization of EqtII in living cells by single molecule imaging. Surprisingly, we found that on the cell surface EqtII did not organize into a unique oligomeric form. Instead, it existed as a mixture of oligomeric species mostly including monomers, dimers, tetramers, and hexamers. Mathematical modeling based on our data supported a new model in which toxin clustering happened in seconds and proceeded via condensation of EqtII dimer units formed upon monomer association. Furthermore, altering the pathway of EqtII assembly strongly affected its toxic activity, which highlights the relevance of the assembly mechanism on toxicity. PMID:25525270

  7. Adsorption of drugs onto a pH responsive poly(N,N-dimethyl aminoethyl methacrylate) grafted anion-exchange membrane in vitro.

    PubMed

    Karppi, Jouni; Akerman, Satu; Akerman, Kari; Sundell, Annika; Nyyssönen, Kristiina; Penttilä, Ilkka

    2007-06-29

    The influence of charge and lipophilicity of acidic and basic model drugs on their adsorption onto poly(N,N-dimethyl aminoethyl methacrylic acid) grafted poly(vinylidene fluoride) (DMAEMA-PVDF) membranes was evaluated. The effect of serum proteins (albumin, IgG) and hormones (cortisol, free thyroxine (T(4)F) and thyrotropin (TSH)) on drug adsorption was also studied. Acidic model drugs (antiepileptics and benzodiazepies) adsorbed to a greater extent onto the membrane from Hepes buffer at ionic strength of 25mM and pH 7.0 than basic drugs (antidepressants) did. Adsorption of acidic model drugs was based on electrostatic interactions between positively charged tertiary amino groups of DMAEMA side-chain and acidic negatively charged drug. Albumin diminished the adsorption of drugs from serum onto the membrane. Lipophilicity was related to the adsorption of acidic model drugs from serum onto the membrane. The degree of grafting had the greatest effect on adsorption of lipophilic drugs, but no influence was observed on adsorption of hydrophilic drugs. The present results showed that acidic drugs and albumin adsorbed onto the membrane, which suggests that the PVDF-DMAEMA membrane may be suitable for separating acidic drugs from protein-free substances for subsequent monitoring and evaluation.

  8. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

    PubMed

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2016-09-10

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Ionic Liquid Confined in Mesoporous Polymer Membrane with Improved Stability for CO2/N2 Separation

    PubMed Central

    Tan, Ming; Lu, Jingting; Zhang, Yang; Jiang, Heqing

    2017-01-01

    Supported ionic liquid membranes (SILMs) have a promising prospect of application in flue gas separation, owing to its high permeability and selectivity of CO2. However, existing SILMs have the disadvantage of poor stability due to the loss of ionic liquid from the large pores of the macroporous support. In this study, a novel SILM with high stability was developed by confining ionic liquid in a mesoporous polymer membrane. First, a mesoporous polymer membrane derived from a soluble, low-molecular-weight phenolic resin precursor was deposited on a porous Al2O3 support, and then 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF4]) was immobilized inside mesopores of phenolic resin, forming the SILM under vacuum. Effects of trans-membrane pressure difference on the SILM separation performance were investigated by measuring the permeances of CO2 and N2. The SILM exhibits a high ideal CO2/N2 selectivity of 40, and an actual selectivity of approximately 25 in a mixed gas (50% CO2 and 50% N2) at a trans-membrane pressure difference of 2.5 bar. Compared to [emim][BF4] supported by polyethersulfone membrane with a pore size of around 0.45 μm, the [emim][BF4] confined in a mesoporous polymer membrane exhibits an improved stability, and its separation performance remained stable for 40 h under a trans-membrane pressure difference of 1.5 bar in a mixed gas before the measurement was intentionally stopped. PMID:28961187

  10. Effect of stabilization temperature during pyrolysis process of P84 co-polyimide-based tubular carbon membrane for H2/N2 and He/N2 separations

    NASA Astrophysics Data System (ADS)

    Sazali, N.; Salleh, W. N. W.; Ismail, A. F.; Ismail, N. H.; Aziz, F.; Yusof, N.; Hasbullah, H.

    2018-04-01

    In this study, the effect of stabilization temperature on the performance of tubular carbon membrane was being investigated. P84 co-polyimide-based tubular carbon membrane will be fabricated through the dip-coating technique. The tubular carbon membrane performance can be controlled by manipulating the pyrolysis conditions which was conducted at different stabilization temperatures of 250, 300, 350, 400, and 450°C under N2 environment (200 ml/min). The prepared membranes were characterized by using scanning electron microscopy (SEM), x-ray diffraction (XRD), and pure gas permeation system. The pure gas of H2, He, and N2 were used to determine the permeation properties of the carbon membrane. The P84 co-polyimide-based tubular carbon membrane stabilized at 300°C demonstrated an excellent permeation property with H2, He, and N2 gas permeance of 1134.51±2.87, 1287.22±2.86 and 2.98±1.28GPU, respectively. The highest H2/N2 and He/N2 selectivity of 380.71±2.34 and 431.95±2.61 was obtained when the stabilization temperature of 450°C was applied. It is concluded that the stabilization temperatures have protrusive effect on the carbon membrane properties specifically their pore structure, and eventually their gas separation properties.

  11. The Evaluations of Hydrogen Permeation and Life Cycle Assessment on Nanocrystallined TiN-BCY Hydrogen Membrane.

    PubMed

    Lee, Soo-Sun; Hong, Tae-Whan

    2016-02-01

    Recently, Membrane technologies are used for the separation of mixtures in various industries. The promising method to reduce the CO2 emission and production of H2 from the coal based power plants is membrane separation with polymer, metal, ceramic and cermet materials. In this study, TiN ceramic material was selected, that is much less expensive than Pd. Also it has resistance to acids and chemically steady. Yttrium doped barium cerate (BCY) is a proton conductor. This perovskite exhibit both high proton conductivity and thermodynamic stability. But its chemical stability is very low under real operating environments. Thus, TiN-BCY may provide'a new membrane material for application. Life cycle assessment (LCA) based on fabrication of membrane and it was carried out to evaluate the energy demand and environmental impact. The analysis is performed according to the recommendations of ISO norms 14040 and obtained using the Gabi 6 software. This LCA will contribute to optimizing the eco-design, reducing the energy consumption and pollutant emissions during the eco-profiles of the TiN-BCY membrane.

  12. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries

    PubMed Central

    Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-01-01

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K+ concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca2+ release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca2+ waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca2+ concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca2+ entry and promote vasoconstriction. PMID:25888510

  13. Al2O3 Disk Supported Si3N4 Hydrogen Purification Membrane for Low Temperature Polymer Electrolyte Membrane Fuel Cells

    PubMed Central

    Liu, Xiaoteng; Christensen, Paul A.; Kelly, Stephen M.; Rocher, Vincent; Scott, Keith

    2013-01-01

    Reformate gas, a commonly employed fuel for polymer electrolyte membrane fuel cells (PEMFCs), contains carbon monoxide, which poisons Pt-containing anodes in such devices. A novel, low-cost mesoporous Si3N4 selective gas separation material was tested as a hydrogen clean-up membrane to remove CO from simulated feed gas to single-cell PEMFC, employing Nafion as the polymer electrolyte membrane. Polarization and power density measurements and gas chromatography showed a clear effect of separating the CO from the gas mixture; the performance and durability of the fuel cell was thereby significantly improved. PMID:24957065

  14. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

    PubMed

    Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

    2016-03-01

    SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. © 2016 Authors; published by Portland Press Limited.

  15. Fabrication of hierarchical porous N-doping carbon membrane by using ;confined nanospace deposition; method for supercapacitor

    NASA Astrophysics Data System (ADS)

    Wang, Guoxu; Liu, Meng; Du, Juan; Liu, Lei; Yu, Yifeng; Sha, Jitong; Chen, Aibing

    2018-03-01

    The membrane carbon materials with hierarchical porous architecture are attractive because they can provide more channels for ion transport and shorten the ions transport path. Herein, we develop a facile way based on "confined nanospace deposition" to fabricate N-dopi-ng three dimensional hierarchical porous membrane carbon material (N-THPMC) via coating the nickel nitrate, silicate oligomers and triblock copolymer P123 on the branches of commercial polyamide membrane (PAM). During high temperature treatment, the mesoporous silica layer and Ni species serve as a "confined nanospace" and catalyst respectively, which are indispensable elements for formation of carbon framework, and the gas-phase carbon precursors which derive from the decomposition of PAM are deposited into the "confined nanospace" forming carbon framework. The N-THPMC with hierarchical macro/meso/microporous structure, N-doping (2.9%) and large specific surface area (994m2 g-1) well inherits the membrane morphology and hierarchical porous structure of PAM. The N-THPMC as electrode without binder exhibits a specific capacitance of 252 F g-1 at the current density of 1 A g-1 in 6 M KOH electrolyte and excellent cycling stability of 92.7% even after 5000 cycles.

  16. Carbonic anhydrase inspired poly(N-vinylimidazole)/zeolite Zn-β hybrid membranes for CO2 capture.

    PubMed

    Liu, Yanni; Wang, Zhi; Shi, Mengqi; Li, Nan; Zhao, Song; Wang, Jixiao

    2018-06-14

    A carbonic anhydrase inspired material was developed by incorporating Zn(ii) ion exchanged zeolite β into poly(N-vinylimidazole) solution. The hydrophobic zeolite channels were designed to imitate the function of the hydrophobic pocket in carbonic anhydrase. The composite membrane prepared by casting the material on a polysulfone ultrafiltration membrane showed a high CO2 permeance of 4620 GPU with a high CO2/N2 selectivity of 224.

  17. The influence of erythrocyte maturity on ion transport and membrane lipid composition in the rat.

    PubMed

    Vokurková, M; Rauchová, H; Dobešová, Z; Loukotová, J; Nováková, O; Kuneš, J; Zicha, J

    2016-01-01

    Significant relationships between ion transport and membrane lipid composition (cholesterol, total phospholipids and sphingomyelins) were found in erythrocytes of salt hypertensive Dahl rats. In these animals mean cellular hemoglobin content correlated negatively with Na(+)-K(+) pump activity and Na(+) leak but positively with Na(+)-K(+) cotransport activity. Immature erythrocytes exhibit lower mean cellular hemoglobin content (MCHC) than mature ones. The aim of the present study was to find a relationship between erythrocyte maturity, membrane lipid composition and ion transport activity in Wistar rats aged three months which were subjected to repeated hemorrhage (blood loss 2 ml/day for 6 days) to enrich circulating erythrocytes with immature forms. Immature and mature erythrocyte fractions in control and hemorrhaged rats were separated by repeated centrifugation. Hemorrhaged rats had increased number of reticulocytes but reduced hematocrit and MCHC compared to control rats. Immature erythrocytes of hemorrhaged rats differed from mature ones of control animals by elevated Na(+)-K(+) pump activity, reduced Na(+)-K(+) cotransport activity and increased Rb(+) leak. These ion transport changes in immature erythrocytes were accompanied by higher concentration of total phospholipids in their cell membranes. Membrane phospholipid content correlated positively with Na(+)-K(+) pump activity and cation leaks but negatively with Na(+)-K(+) cotransport activity. Moreover, they were also negatively related with MCHC which correlated negatively with Na(+)-K(+) pump activity and Rb(+) leak but positively with Na(+)-K(+) cotransport activity. Thus certain abnormalities of erythrocyte ion transport and membrane lipid composition detected in hypertensive animals might be caused by higher incidence of immature cells.

  18. N-Doped TiO₂-Coated Ceramic Membrane for Carbamazepine Degradation in Different Water Qualities.

    PubMed

    Luster, Enbal; Avisar, Dror; Horovitz, Inna; Lozzi, Luca; Baker, Mark A; Grilli, Rossana; Mamane, Hadas

    2017-07-31

    The photocatalytic degradation of the model pollutant carbamazepine (CBZ) was investigated under simulated solar irradiation with an N-doped TiO₂-coated Al₂O₃ photocatalytic membrane, using different water types. The photocatalytic membrane combines photocatalysis and membrane filtration in a single step. The impact of each individual constituent such as acidity, alkalinity, dissolved organic matter (DOM), divalent cations (Mg 2+ and Ca 2+ ), and Cl - on the degradation of CBZ was examined. CBZ in water was efficiently degraded by an N-doped TiO₂-coated Al₂O₃ membrane. However, elements added to the water, which simulate the constituents of natural water, had an impact on the CBZ degradation. Water alkalinity inhibited CBZ degradation mostly due to increase in pH while radical scavenging by carbonate was more dominant at higher values (>200 mg/L as CaCO₃). A negative effect of Ca 2+ addition on photocatalytic degradation was found only in combination with phosphate buffer, probably caused by deposition of CaHPO₄ or CaHPO₄·2H₂O on the catalyst surface. The presence of Cl - and Mg 2+ ions had no effect on CBZ degradation. DOM significantly inhibited CBZ degradation for all tested background organic compounds. The photocatalytic activity of N-doped TiO₂-coated Al₂O₃ membranes gradually decreased after continuous use; however, it was successfully regenerated by 0.1% HCl chemical cleaning. Nevertheless, dissolution of metals like Al and Ti should be monitored following acid cleaning.

  19. IroN, a Novel Outer Membrane Siderophore Receptor Characteristic of Salmonella enterica

    PubMed Central

    Bäumler, Andreas J.; Norris, Tracy L.; Lasco, Todd; Voigt, Wolfgang; Reissbrodt, Rolf; Rabsch, Wolfgang; Heffron, Fred

    1998-01-01

    Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize iroN, a gene located downstream of the iroBC operon in the iroA locus of Salmonella enterica serotype Typhi. Like iroBC, the iroN gene is present in all phylogenetic lineages of S. enterica but is absent from closely related species such as Salmonella bongori or Escherichia coli. Comparison of the deduced amino acid sequence of iroN with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in S. enterica and expression in E. coli identified a 78-kDa outer membrane protein as the iroN gene product. When introduced into an E. coli fepA cir fiu aroB mutant on a cosmid, iroN mediated utilization of structurally related catecholate siderophores, including N-(2,3-dihydroxybenzoyl)-l-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-l-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-l-lysine amide, and enterochelin. These results suggest that the iroA locus functions in iron acquisition in S. enterica. PMID:9515912

  20. Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

    DOE PAGES

    Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

  1. Contribution of glycerophospholipids and sphingomyelin to the circulating NEFA.

    PubMed

    Maier, Felicitas; Demmelmair, Hans; Fugmann, Marina; Hellmuth, Christian; Lechner, Andreas; Koletzko, Berthold; Uhl, Olaf

    2016-07-01

    Serum nonesterified fatty acids (NEFA) are known to be associated with the development of insulin resistance. Recently, differences in the NEFA profile were found in subjects with history of gestational diabetes (postGDM) and healthy controls. Little is known about the NEFA sources in the postprandial state, which prevails most of the day in humans in modern societies. In the present study, we aimed to explore the potential contributions of glycerophospholipid (GPL) and sphingomyelin (SM) fatty acids to the circulating NEFA. Serum-samples of 19 postGDM women and 20 controls were obtained in fasting state (t0) and 90 minutes (t90) after an oral glucose tolerance test. Fatty acid composition of NEFA and SM were analyzed with liquid chromatography coupled to triple quadrupole mass spectrometry and GPL by gas chromatography. The ratio of individual NEFA at t90 vs. t0 (t90/0-ratio) showed no difference between the two groups but increased with chain-length (7% for C16:1, 82% for C26:3). Only NEFA 10:0 was found with lower concentration at t0 and t90 in postGDM. At t90, long-chain polyunsaturated fatty acid correlated closely between NEFA and GPL in postGDM (20:5, 22:4, 22:5 and 22:6) and controls (20:3, 20:4 and 20:5). Very long-chain fatty acid 24:0 correlated significantly between NEFA and SM in postGDM and controls. Saturated and monounsaturated fatty acids correlated less between NEFA and GPL or SM. The NEFA composition varied highly between fasting and fed state in both groups. GPL appeared to contribute long-chain polyunsaturated fatty acid, while SM appeared to contribute very long-chain fatty acids to the NEFA pool. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-beta-cyclodextrin. Role of low-density caveolae-like membranes.

    PubMed

    Buschiazzo, Jorgelina; Bonini, Ida C; Alonso, Telma S

    2008-06-01

    The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5'nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-beta-cyclodextrin (MbetaCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MbetaCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.

  3. Fabrication of high-quality superconductor-insulator-superconductor junctions on thin SiN membranes

    NASA Technical Reports Server (NTRS)

    Garcia, Edouard; Jacobson, Brian R.; Hu, Qing

    1993-01-01

    We have successfully fabricated high-quality and high-current density superconductor-insulator-superconductor (SIS) junctions on freestanding thin silicon nitride (SIN) membranes. These devices can be used in a novel millimeter-wave and THz receiver system which is made using micromachining. The SIS junctions with planar antennas were fabricated first on a silicon wafer covered with a SiN membrane, the Si wafer underneath was then etched away using an anisotropic KOH etchant. The current-voltage characteristics of the SIS junctions remained unchanged after the whole process, and the junctions and the membrane survived thermal cycling.

  4. Synergistic antioxidant activity of milk sphingomyeline and its sphingoid base with α-tocopherol on fish oil triacylglycerol.

    PubMed

    Shimajiri, Junki; Shiota, Makoto; Hosokawa, Masashi; Miyashita, Kazuo

    2013-08-21

    The effects of milk phospholipids (PLs), sphingolipids (SLs), and their sphingoid backbone on the oxidation of fish oil triacylglycerol (TAG) were examined with or without α-tocopherol. All compounds had little effect on the TAG oxidation in the absence of α-tocopherol. On the other hand, they could act synergistically with α-tocopherol. The highest synergistic activity was shown by sphingoid bases, followed by sphingomyelin (SPM) and other amine-containing PLs and SLs. This result showed that the synergistic activity increased with an increasing concentration of amine group of PLs, SLs, or sphingoid bases in the reaction mixture. The comparison of changes in α-tocopherol content in fish oil TAG and tricaprylin suggested that antioxidant compounds would be formed from the amine group and the lipid oxidation products in a mild oxidation condition controlled by α-tocopherol.

  5. Modifications in plasma membrane lipid composition and morphological features of AH-130 hepatoma cells by polyenylphosphatidylcholine in vivo treatment.

    PubMed

    Cinosi, Vincenzo; Antonini, Roberto; Crateri, Pasqualina; Arancia, Giuseppe

    2011-07-01

    The plasma membrane lipid composition in AH-130 hepatoma cells was found to change remarkably after polyenylphosphatidylcholine (PPC) treatment. Plasma membranes from cells grown in rats treated for 7 days i.v. with 20 mg/kg/day PPC, when compared to those of control cells, did not show significantly different amounts of cholesterol or phospholipids relative to protein content, but, surprisingly, the individual phospholipid distribution inside the two membrane leaflets changed dramatically. Phosphatidylcholine (PC), the major phospholipid in the external membrane leaflet, increased ~47% (p<0.001). By contrast, phosphatidylethanolamine (PE), the most important component of the inner leaflet, decreased nearly 37% (p<0.001), while sphingomyelin (SM) also decreased ~17%, (p=0.1). Tumor cells collected from control rats at the same time interval and observed by scanning electron microscopy, exhibited a spherical shape with numerous and randomly distributed long microvilli, the same morphological and ultrastructural features displayed by the implanted cells. Conversely, tumor cells from PPC-treated rats no longer showed the roundish cell profile, and microvilli appeared shortened and enlarged, with the formation of surface blebs. Transmission electron microscopy observations confirmed the morphological and ultrastructural cell changes, mainly seen as loss of microvilli and intense cytoplasmic vacuolization. Taken together, these results indicate that the new phospholipid class distribution in the plasma membrane leaflets, modifying tumor cell viable structures, produced heavy cell damage and in many cases brought about complete cellular disintegration.

  6. Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr-/- Mice.

    PubMed

    Pollock, Abigail H; Tedla, Nicodemus; Hancock, Sarah E; Cornely, Rhea; Mitchell, Todd W; Yang, Zhengmin; Kockx, Maaike; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

    2016-05-15

    Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4(+) and CD8(+) T cell proliferation and resulted in an increased proportion of CD4(+) central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment. Copyright © 2016 by The American Association of Immunologists, Inc.

  7. Biological treatment of mixtures of toluene and n-hexane vapours in a hollow fibre membrane bioreactor.

    PubMed

    Zhao, Kang; Xiu, Guangli; Xu, Lihang; Zhang, Danian; Zhang, Xiaofeng; Deshusses, Marc A

    2011-04-01

    Membrane bioreactors are gaining interest for the control of contaminated air streams. In this study, the removal of toluene and n-hexane vapours in a hollow fibre membrane bioreactor (HFMB) was investigated. The focus was on quantifying the possible interactions occurring during the simultaneous biotreatment of the two volatile pollutants. Two lab-scale units fitted with microporous polypropylene hollow fibre membranes were connected in series and inoculated with activated sludge. Contaminated air was passed through the lumen at gas residence times ranging from 2.3 to 9.4 s while a pollutant-degrading biofilm developed on the shell side of the fibres. When toluene was treated alone, very high elimination capacities (up to 750 g m(-3) h(-1) based on lumen volume, or 1.25 g m(-2) h(-1) when normalized by the hollow fibre membrane area) were reached. When toluene and hexane were treated simultaneously, toluene biodegradation was partially inhibited by n-hexane, resulting in lower toluene removal rates. On the other hand, hexane removal was only marginally affected by the presence of toluene and was degraded at very high rates (upwards of 440 g m(-3) h(-1) or 0.73 g m(-2) h(-1) without breakthrough). Overall, this study demonstrates that mixtures of toluene and n-hexane vapours can be effectively removed in hollow fibre membrane bioreactors and that complex biological interactions may affect one or more of the pollutants undergoing treatment in gas-phase membrane bioreactors.

  8. Sphingomyelin phosphodiesterase-1 (SMPD1) coding variants do not contribute to low levels of high-density lipoprotein cholesterol

    PubMed Central

    Dastani, Zari; Ruel, Isabelle L; Engert, James C; Genest, Jacques; Marcil, Michel

    2007-01-01

    Background Niemann-Pick disease type A and B is caused by a deficiency of acid sphingomyelinase due to mutations in the sphingomyelin phosphodiesterase-1 (SMPD1) gene. In Niemann-Pick patients, SMPD1 gene defects are reported to be associated with a severe reduction in plasma high-density lipoprotein (HDL) cholesterol. Methods Two common coding polymorphisms in the SMPD1 gene, the G1522A (G508R) and a hexanucleotide repeat sequence within the signal peptide region, were investigated in 118 unrelated subjects of French Canadian descent with low plasma levels of HDL-cholesterol (< 5th percentile for age and gender-matched subjects). Control subjects (n = 230) had an HDL-cholesterol level > the 25th percentile. Results For G1522A the frequency of the G and A alleles were 75.2% and 24.8% respectively in controls, compared to 78.6% and 21.4% in subjects with low HDL-cholesterol (p = 0.317). The frequency of 6 and 7 hexanucleotide repeats was 46.2% and 46.6% respectively in controls, compared to 45.6% and 49.1% in subjects with low HDL-cholesterol (p = 0.619). Ten different haplotypes were observed in cases and controls. Overall haplotype frequencies in cases and controls were not significantly different. Conclusion These results suggest that the two common coding variants at the SMPD1 gene locus are not associated with low HDL-cholesterol levels in the French Canadian population. PMID:18088425

  9. Chronic Ethanol Consumption Profoundly Alters Regional Brain Ceramide and Sphingomyelin Content in Rodents

    PubMed Central

    2015-01-01

    Ceramides (CER) are involved in alcohol-induced neuroinflammation. In a mouse model of chronic alcohol exposure, 16 CER and 18 sphingomyelin (SM) concentrations from whole brain lipid extracts were measured using electrospray mass spectrometry. All 18 CER concentrations in alcohol exposed adults increased significantly (range: 25–607%); in juveniles, 6 CER decreased (range: −9 to −37%). In contrast, only three SM decreased in adult and one increased significantly in juvenile. Next, regional identification at 50 μm spatial resolution from coronal sections was obtained with matrix implanted laser desorption/ionization mass spectrometry imaging (MILDI-MSI) by implanting silver nanoparticulate matrices followed by focused laser desorption. Most of the CER and SM quantified in whole brain extracts were detected in MILDI images. Coronal sections from three brain levels show qualitative regional changes in CER-SM ion intensities, as a function of group and brain region, in cortex, striatum, accumbens, habenula, and hippocampus. Highly correlated changes in certain white matter CER-SM pairs occur in regions across all groups, including the hippocampus and the lateral (but not medial) cerebellar cortex of adult mice. Our data provide the first microscale MS evidence of regional lipid intensity variations induced by alcohol. PMID:25387107

  10. Kinetic Defects Induced by Melittin in Model Lipid Membranes: A Solution Atomic Force Microscopy Study.

    PubMed

    Pan, Jianjun; Khadka, Nawal K

    2016-05-26

    Quantitative characterization of membrane defects (pores) is important for elucidating the molecular basis of many membrane-active peptides. We study kinetic defects induced by melittin in vesicular and planar lipid bilayers. Fluorescence spectroscopy measurements indicate that melittin induces time-dependent calcein leakage. Solution atomic force microscopy (AFM) is used to visualize melittin-induced membrane defects. After initial equilibration, the most probable defect radius is ∼3.8 nm in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) bilayers. Unexpectedly, defects become larger with longer incubation, accompanied by substantial shape transformation. The initial defect radius is ∼4.7 nm in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. Addition of 30 mol % cholesterol to DOPC bilayers suppresses defect kinetics, although the inhibitory impact is negated by longer incubation. Overall, the kinetic rate of defect development follows DLPC > DOPC > DOPC/cholesterol. Kinetic defects are also observed when anionic lipids are present. Based on the observation that defects can occupy as large as 40% of the bilayer surface, we propose a kinetic defect growth model. We also study the effect of melittin on the phase behavior of DOPC/egg-sphingomyelin/cholesterol bilayers. We find that melittin initially suppresses or eliminates liquid-ordered (Lo) domains; Lo domains gradually emerge and become the dominant species with longer incubation; and defects in phase-coexisting bilayers have a most probable radius of ∼5 nm and are exclusively localized in the liquid-disordered (Ld) phase. Our experimental data highlight that melittin-induced membrane defects are not static; conversely, spontaneous defect growth is intrinsically associated with membrane permeabilization exerted by melittin.

  11. N-Doped TiO2-Coated Ceramic Membrane for Carbamazepine Degradation in Different Water Qualities

    PubMed Central

    Luster, Enbal; Avisar, Dror; Horovitz, Inna; Lozzi, Luca; Baker, Mark A.; Grilli, Rossana; Mamane, Hadas

    2017-01-01

    The photocatalytic degradation of the model pollutant carbamazepine (CBZ) was investigated under simulated solar irradiation with an N-doped TiO2-coated Al2O3 photocatalytic membrane, using different water types. The photocatalytic membrane combines photocatalysis and membrane filtration in a single step. The impact of each individual constituent such as acidity, alkalinity, dissolved organic matter (DOM), divalent cations (Mg2+ and Ca2+), and Cl− on the degradation of CBZ was examined. CBZ in water was efficiently degraded by an N-doped TiO2-coated Al2O3 membrane. However, elements added to the water, which simulate the constituents of natural water, had an impact on the CBZ degradation. Water alkalinity inhibited CBZ degradation mostly due to increase in pH while radical scavenging by carbonate was more dominant at higher values (>200 mg/L as CaCO3). A negative effect of Ca2+ addition on photocatalytic degradation was found only in combination with phosphate buffer, probably caused by deposition of CaHPO4 or CaHPO4·2H2O on the catalyst surface. The presence of Cl− and Mg2+ ions had no effect on CBZ degradation. DOM significantly inhibited CBZ degradation for all tested background organic compounds. The photocatalytic activity of N-doped TiO2-coated Al2O3 membranes gradually decreased after continuous use; however, it was successfully regenerated by 0.1% HCl chemical cleaning. Nevertheless, dissolution of metals like Al and Ti should be monitored following acid cleaning. PMID:28758982

  12. ABC-transporters are localized in caveolin-1-positive and reggie-1-negative and reggie-2-negative microdomains of the canalicular membrane in rat hepatocytes.

    PubMed

    Ismair, Manfred G; Häusler, Stephanie; Stuermer, Claudia A; Guyot, Christelle; Meier, Peter J; Roth, Jürgen; Stieger, Bruno

    2009-05-01

    The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4 degrees C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: "Lubrol-microdomains" and "Triton-microdomains". "Lubrol-microdomains" contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion.

  13. N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

    PubMed

    Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

    2009-03-23

    N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

  14. High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

    PubMed Central

    Sot, Jesús; Manni, Marco M.; Viguera, Ana R.; Castañeda, Verónica; Cano, Ainara; Alonso, Cristina; Gil, David; Valle, Mikel; Alonso, Alicia; Goñi, Félix M.

    2014-01-01

    The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition. PMID:25517149

  15. 21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

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

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

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

  16. Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

    PubMed

    Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

    2011-06-17

    To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

  17. Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

    PubMed Central

    Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

    2011-01-01

    To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

  18. Nonadditive Compositional Curvature Energetics of Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Sodt, A. J.; Venable, R. M.; Lyman, E.; Pastor, R. W.

    2016-09-01

    The unique properties of the individual lipids that compose biological membranes together determine the energetics of the surface. The energetics of the surface, in turn, govern the formation of membrane structures and membrane reshaping processes, and thus they will underlie cellular-scale models of viral fusion, vesicle-dependent transport, and lateral organization relevant to signaling. The spontaneous curvature, to the best of our knowledge, is always assumed to be additive. We describe observations from simulations of unexpected nonadditive compositional curvature energetics of two lipids essential to the plasma membrane: sphingomyelin and cholesterol. A model is developed that connects molecular interactions to curvature stress, and which explains the role of local composition. Cholesterol is shown to lower the number of effective Kuhn segments of saturated acyl chains, reducing lateral pressure below the neutral surface of bending and favoring positive curvature. The effect is not observed for unsaturated (flexible) acyl chains. Likewise, hydrogen bonding between sphingomyelin lipids leads to positive curvature, but only at sufficient concentration, below which the lipid prefers negative curvature.

  19. Ceramide signaling in cancer and stem cells

    PubMed Central

    Bieberich, Erhard

    2008-01-01

    Most of the previous work on the sphingolipid ceramide has been devoted to its function as an apoptosis inducer. Recent studies, however, have shown that in stem cells, ceramide has additional nonapoptotic functions. In this article, ceramide signaling will be reviewed in light of ‘systems interface biology’: as an interconnection of sphingolipid metabolism, membrane biophysics and cell signaling. The focus will be on the metabolic interconversion of ceramide and sphingomyelin or sphingosine-1-phosphate. Lipid rafts and sphingolipid-induced protein scaffolds will be discussed as a membrane interface for lipid-controlled cell signaling. Ceramide/sphingomyelin and ceramide/sphingosine-1-phosphate-interdependent cell-signaling pathways are significant for the regulation of cell polarity, apoptosis and/or proliferation, and as novel pharmacologic targets in cancer and stem cells. PMID:19050750

  20. Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility

    NASA Astrophysics Data System (ADS)

    Schmitz, Gerd; Goetz, Alexandra; Orso, Evelyn; Rothe, Gregor

    1998-04-01

    The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C6-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C6-NBD-sphingolyelin and C6-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport.

  1. Morphological differentiation of N1E-115 neuroblastoma cells by dimethyl sulfoxide activation of lipid second messengers.

    PubMed

    Clejan, S; Dotson, R S; Wolf, E W; Corb, M P; Ide, C F

    1996-04-10

    Quantitative changes in the lipid second messenger diacylglycerol (DAG) were studied in the rat neuroblastoma N1E-115 following exposure to the differentiating agent dimethylsulfoxide (DMSO). Relatively high basal levels of DAG are present in these cells, and addition of 2% DMSO elicited a biphasic increase in DAG levels, dependent on the presence of extracellular Ca2+. Exposure to DMSO also elicited a rapid increase in inositol phosphate and a slight increase in phosphatidic acid (PA), trailing that of DAG. The molecular species (MS) of DAG were analyzed. Within 60 s of DMSO application there were transient increases of DAG representative of phosphatidylinositol (PI) hydrolysis. At longer intervals, more DAG originated from phosphatidylcholine. The MS composition of newly formed PA resembled that of PI and native DAG. Inhibition studies indicated that DAG is formed in the DMSO-treated cells by phospholipases C and that PA formed later is a result of DAG phosphorylation and not activity of phospholipase D (PLD). Undifferentiated cells exhibited an active PLD pathway. In contrast, PLD in DMSO-differentiated cells was not active. In examining the involvement of the sphingomyelin pathway, DMSO exposure was found to increase ceramide levels with a concomitant decrease in sphingomyelin. Addition of the exogenous, soluble analog C6-ceramide to undifferentiated cells resulted in dramatic reductions in DAG and PA levels and PLD activity. These results indicate that DMSO treatment inactivates PLD while activating phospholipases C and the sphingomyelin pathway, suggesting a "switch" between signal transduction pathways in the undifferentiated and differentiated states of N1E-115.

  2. Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells

    PubMed Central

    2013-01-01

    Background Some of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms. Methods Eight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model. Results PPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-β-cyclodextrin (MβCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo. Conclusions This study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies. PMID:23889969

  3. Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

    PubMed

    Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

    2018-02-01

    Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts

    PubMed Central

    Schenkel, Laila C.; Singh, Ratnesh K.; Michel, Vera; Zeisel, Steven H.; da Costa, Kerry-Ann; Johnson, Amy R.; Mudd, Harvey S.; Bakovic, Marica

    2015-01-01

    Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2–3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.—Schenkel, L. C., Singh, R. K., Michel, V., Zeisel, S. H., da Costa, K.-A., Johnson, A. R., Mudd, H. S., Bakovic, M. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts. PMID:25466896

  5. N-terminal-mediated oligomerization of DnaA drives the occupancy-dependent rejuvenation of the protein on the membrane.

    PubMed

    Aranovich, Alexander; Braier-Marcovitz, Shani; Ansbacher, Esti; Granek, Rony; Parola, Abraham H; Fishov, Itzhak

    2015-08-13

    DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once per cell division cycle. Its overall activity cycle is driven by ATP hydrolysis and ADP-ATP exchange. The latter can be promoted by binding to specific sequences on the chromosome and/or to acidic phospholipids in the membrane. We have previously shown that the transition into an active form (rejuvenation) is strongly co-operative with respect to DnaA membrane occupancy. Only at low membrane occupancy is DnaA reactivation efficiently catalysed by the acidic phospholipids. The present study was aimed at unravelling the molecular mechanism underlying the occupancy-dependent DnaA rejuvenation. We found that truncation of the DnaA N-terminal completely abolishes the co-operative transformation between the high and low occupancy states (I and II respectively) without affecting the membrane binding. The environmentally sensitive fluorophore specifically attached to the N-terminal cysteines of DnaA reported on occupancy-correlated changes in its vicinity. Cross-linking of DnaA with a short homobifunctional reagent revealed that state II of the protein on the membrane corresponds to a distinct oligomeric form of DnaA. The kinetic transition of DnaA on the membrane surface is described in the present study by a generalized 2D condensation phase transition model, confirming the existence of two states of DnaA on the membrane and pointing to the possibility that membrane protein density serves as an on-off switch in vivo. We conclude that the DnaA conformation attained at low surface density drives its N-terminal-mediated oligomerization, which is presumably a pre-requisite for facilitated nt exchange. © 2015 Authors.

  6. External push and internal pull forces recruit curvature sensing N-BAR domain proteins to the plasma membrane

    PubMed Central

    Galic, Milos; Jeong, Sangmoo; Tsai, Feng-Chiao; Joubert, Lydia-Marie; Wu, Yi I.; Hahn, Klaus M.; Cui, Yi; Meyer, Tobias

    2012-01-01

    Many of the more than 20 mammalian proteins with N-BAR domains1-2 control cell architecture3 and endocytosis4-5 by associating with curved sections of the plasma membrane (PM)6. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the PM or indirectly by PM-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally-induced inward deformation of the PM by cone-shaped nanostructures (Nanocones) and internally-induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved PM. Markedly, live-cell imaging in adherent cells showed selective recruitment of full length N-BAR proteins and isolated N-BAR domains to PM sub-regions above Nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by Nanocones. We further showed that N-BAR domains are periodically recruited to curved PM sites during local lamellipodia retraction in the front of migrating cells. Recruitment required Myosin II-generated force applied to PM connected actin cables. Together, our study shows that N-BAR domains can be directly recruited to the PM by external push or internal pull forces that locally curve the PM. PMID:22750946

  7. Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

    PubMed

    Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

    2017-02-23

    Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

  8. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts.

    PubMed

    Schenkel, Laila C; Singh, Ratnesh K; Michel, Vera; Zeisel, Steven H; da Costa, Kerry-Ann; Johnson, Amy R; Mudd, Harvey S; Bakovic, Marica

    2015-05-01

    Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired. © FASEB.

  9. Impact of Lipid Composition and Receptor Conformation on the Spatio-temporal Organization of μ-Opioid Receptors in a Multi-component Plasma Membrane Model

    PubMed Central

    Marino, Kristen A.; Prada-Gracia, Diego; Provasi, Davide; Filizola, Marta

    2016-01-01

    The lipid composition of cell membranes has increasingly been recognized as playing an important role in the function of various membrane proteins, including G Protein-Coupled Receptors (GPCRs). For instance, experimental and computational evidence has pointed to lipids influencing receptor oligomerization directly, by physically interacting with the receptor, and/or indirectly, by altering the bulk properties of the membrane. While the exact role of oligomerization in the function of class A GPCRs such as the μ-opioid receptor (MOR) is still unclear, insight as to how these receptors oligomerize and the relevance of the lipid environment to this phenomenon is crucial to our understanding of receptor function. To examine the effect of lipids and different MOR conformations on receptor oligomerization we carried out extensive coarse-grained molecular dynamics simulations of crystal structures of inactive and/or activated MOR embedded in an idealized mammalian plasma membrane composed of 63 lipid types asymmetrically distributed across the two leaflets. The results of these simulations point, for the first time, to specific direct and indirect effects of the lipids, as well as the receptor conformation, on the spatio-temporal organization of MOR in the plasma membrane. While sphingomyelin-rich, high-order lipid regions near certain transmembrane (TM) helices of MOR induce an effective long-range attractive force on individual protomers, both long-range lipid order and interface formation are found to be conformation dependent, with a larger number of different interfaces formed by inactive MOR compared to active MOR. PMID:27959924

  10. Theoretical investigation of Lamb wave characteristics in AlN/3C-SiC composite membranes

    NASA Astrophysics Data System (ADS)

    Lin, Chih-Ming; Chen, Yung-Yu; Pisano, Albert P.

    2010-11-01

    Cubic silicon carbide (3C-SiC) layer can provide advantages of high frequency and high quality factor for Lamb wave devices due to the superior properties of high acoustic velocity and low acoustic loss. In this study, Lamb wave propagation characteristics in composite membranes consisting of a c-axis oriented aluminum nitride (AlN) film and an epitaxial 3C-SiC (100) layer are investigated by theoretical calculation. The lowest symmetric mode Lamb wave propagating along the [011] direction exhibits a phase velocity higher than 10 000 m/s and an electromechanical coupling coefficient above 2% in the AlN/3C-SiC multilayered membranes.

  11. The preference of tryptophan for membrane interfaces: insights from N-methylation of tryptophans in gramicidin channels.

    PubMed

    Sun, Haiyan; Greathouse, Denise V; Andersen, Olaf S; Koeppe, Roger E

    2008-08-08

    To better understand the structural and functional roles of tryptophan at the membrane/water interface in membrane proteins, we examined the structural and functional consequences of Trp --> 1-methyl-tryptophan substitutions in membrane-spanning gramicidin A channels. Gramicidin A channels are miniproteins that are anchored to the interface by four Trps near the C terminus of each subunit in a membrane-spanning dimer. We masked the hydrogen bonding ability of individual or multiple Trps by 1-methylation of the indole ring and examined the structural and functional changes using circular dichroism spectroscopy, size exclusion chromatography, solid state (2)H NMR spectroscopy, and single channel analysis. N-Methylation causes distinct changes in the subunit conformational preference, channel-forming propensity, single channel conductance and lifetime, and average indole ring orientations within the membrane-spanning channels. The extent of the local ring dynamic wobble does not increase, and may decrease slightly, when the indole NH is replaced by the non-hydrogen-bonding and more bulky and hydrophobic N-CH(3) group. The changes in conformational preference, which are associated with a shift in the distribution of the aromatic residues across the bilayer, are similar to those observed previously with Trp --> Phe substitutions. We conclude that indole N-H hydrogen bonding is of major importance for the folding of gramicidin channels. The changes in ion permeability, however, are quite different for Trp --> Phe and Trp --> 1-methyl-tryptophan substitutions, indicating that the indole dipole moment and perhaps also ring size and are important for ion permeation through these channels.

  12. Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

    PubMed Central

    Chen, C S; Martin, O C; Pagano, R E

    1997-01-01

    N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from

  13. Yak milk fat globules from the Qinghai-Tibetan Plateau: Membrane lipid composition and morphological properties.

    PubMed

    Luo, Jie; Huang, Ziyu; Liu, Hongna; Zhang, Yan; Ren, Fazheng

    2018-04-15

    Yak milk fat products constitute the base of Qinghai-Tibetan pastoralists' daily food intake. Despite the great importance of fat in processing and pastoralists' health, studies about yak milk fat are scarce. In this study, the lipid composition and the morphological properties of milk fat globule membranes (MFGMs) of yak milk were investigated. The results demonstrated that the yak milk had a higher cholesterol and sphingomyelin content compared to cow milk. In situ structural investigations performed at 25 °C by confocal microscopy showed the presence of lipid domains in yak MFGM, with a larger number and wider size range compared to cow milk. Moreover, the simultaneous localization of glycosylated molecules and polar lipids indicated that glycosylated molecules could be integrated into the lipid domains in yak MFGM. Different characteristics in yak MFGM could be related to the lipid composition and may affect the functions of yak milk lipids during processing and digestion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Effect of C/N shock variation on the performances of a moving bed membrane bioreactor.

    PubMed

    Di Trapani, Daniele; Di Bella, Gaetano; Mannina, Giorgio; Torregrossa, Michele; Viviani, Gaspare

    2015-08-01

    The effect of a sharp variation of C/N ratio in a moving bed membrane bioreactor (MB-MBR) pilot plant treating high strength wastewater has been investigated. The experimental campaign was divided into two periods, each characterized by a different C/N ratio (namely, 2.5 and 15, Period 1 and Period 2, respectively). The MB-MBR system was analyzed in terms of organic carbon removal, nitrification efficiency, biokinetic activity and fouling behavior. The results showed that the nitrification process was severely affected by lower C/N value and by high concentration of ammonia. It was noticed an extensive stress effect on the autotrophic bacteria. Furthermore, it was observed an increase of the resistance related to particle deposition into membrane pores, likely due to a worsening of the cake layer features, with a reduction of the "pre-filter" effect, also related to the increase of the total Extracellular Polymeric Substances production with the C/N ratio. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

    PubMed

    Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-03-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

  16. Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells.

    PubMed

    Kouzel, Ivan U; Pohlentz, Gottfried; Storck, Wiebke; Radamm, Lena; Hoffmann, Petra; Bielaszewska, Martina; Bauwens, Andreas; Cichon, Christoph; Schmidt, M Alexander; Mormann, Michael; Karch, Helge; Müthing, Johannes

    2013-03-01

    Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.

  17. Effects of chronic N fertilization on foliar membranes, cold tolerance, and carbon storage in montane red spruce

    Treesearch

    Paul G. Schaberg; Donald H. DeHayes; Gary J. Hawley; Paula F. Murakami; G. Richard Strimbeck; Steven G. McNulty

    2002-01-01

    We evaluated the influence of protracted low-level nitrogen (N) fertilization on foliar membrane-associated calcium (mCa), sugar and starch concentrations, membrane stability, winter cold tolerance, and freezing injury of red spruce (Picea rubens Sarg.) trees growing in six experimental plots on Mount Ascutney, Vermont. For 12 consecutive years...

  18. Effect of the nature of phospholipids on the degree of their interaction with isobornylphenol antioxidants

    NASA Astrophysics Data System (ADS)

    Marakulina, K. M.; Kramor, R. V.; Lukanina, Yu. K.; Plashchina, I. G.; Polyakov, A. V.; Fedorova, I. V.; Chukicheva, I. Yu.; Kutchin, A. V.; Shishkina, L. N.

    2016-02-01

    The parameters of complexation between natural phospholipids (lecithin, sphingomyelin, and cephalin) with antioxidants of a new class, isobornylphenols (IBPs), were determined by UV and IR spectroscopy. The self-organization of phospholipids (PLs) was studied depending on the structure of IBPs by dynamic light scattering. The nature of phospholipids and the structure of IBPs was found to produce a substantial effect both on the degree of complexation and on the size of PL aggregates in a nonpolar solvent. Based on the obtained data it was concluded that the structure of biological membranes mainly depends on the complexation of IBP with sphingomyelin.

  19. Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif

    PubMed Central

    Wang, Yaofeng; Kraut, Rachel; Mu, Yuguang

    2015-01-01

    The Amyloid-β (Aβ)-derived, sphingolipid binding domain (SBD) peptide is a fluorescently tagged probe used to trace the diffusion behavior of sphingolipid-containing microdomains in cell membranes through binding to a constellation of glycosphingolipids, sphingomyelin, and cholesterol. However, the molecular details of the binding mechanism between SBD and plasma membrane domains remain unclear. Here, to investigate how the peptide recognizes the lipid surface at an atomically detailed level, SBD peptides in the environment of raft-like bilayers were examined in micro-seconds-long molecular dynamics simulations. We found that SBD adopted a coil-helix-coil structural motif, which binds to multiple GT1b gangliosides via salt bridges and CH–π interactions. Our simulation results demonstrate that the CH–π and electrostatic forces between SBD monomers and GT1b gangliosides clusters are the main driving forces in the binding process. The presence of the fluorescent dye and linker molecules do not change the binding mechanism of SBD probes with gangliosides, which involves the helix-turn-helix structural motif that was suggested to constitute a glycolipid binding domain common to some sphingolipid interacting proteins, including HIV gp120, prion, and Aβ. PMID:26540054

  20. Hybrid GaN LED with capillary-bonded II-VI MQW color-converting membrane for visible light communications

    NASA Astrophysics Data System (ADS)

    Santos, Joao M. M.; Jones, Brynmor E.; Schlosser, Peter J.; Watson, Scott; Herrnsdorf, Johannes; Guilhabert, Benoit; McKendry, Jonathan J. D.; De Jesus, Joel; Garcia, Thor A.; Tamargo, Maria C.; Kelly, Anthony E.; Hastie, Jennifer E.; Laurand, Nicolas; Dawson, Martin D.

    2015-03-01

    The rapid emergence of gallium-nitride (GaN) light-emitting diodes (LEDs) for solid-state lighting has created a timely opportunity for optical communications using visible light. One important challenge to address this opportunity is to extend the wavelength coverage of GaN LEDs without compromising their modulation properties. Here, a hybrid source for emission at 540 nm consisting of a 450 nm GaN micro-sized LED (micro-LED) with a micron-thick ZnCdSe/ZnCdMgSe multi-quantum-well color-converting membrane is reported. The membrane is liquid-capillary-bonded directly onto the sapphire window of the micro-LED for full hybridization. At an injection current of 100 mA, the color-converted power was found to be 37 μW. At this same current, the -3 dB optical modulation bandwidth of the bare GaN and hybrid micro-LEDs were 79 and 51 MHz, respectively. The intrinsic bandwidth of the color-converting membrane was found to be power-density independent over the range of the micro-LED operation at 145 MHz, which corresponds to a mean carrier lifetime of 1.9 ns.

  1. Laser ablation for membrane processing of AlGaN/GaN- and micro structured ferroelectric thin film MEMS and SiC pressure sensors for extreme conditions

    NASA Astrophysics Data System (ADS)

    Zehetner, J.; Vanko, G.; Dzuba, J.; Ryger, I.; Lalinsky, T.; Benkler, Manuel; Lucki, Michal

    2015-05-01

    AlGaN/GaN based high electron mobility transistors (HEMTs), Schottky diodes and/or resistors have been presented as sensing devices for mechanical or chemical sensors operating in extreme conditions. In addition we investigate ferroelectric thin films for integration into micro-electro-mechanical-systems (MEMS). Creation of appropriate diaphragms and/or cantilevers out of SiC is necessary for further improvement of sensing properties of such MEMS sensors. For example sensitivity of the AlGaN/GaN based MEMS pressure sensor can be modified by membrane thickness. We demonstrated that a 4H-SiC 80μm thick diaphragms can be fabricated much faster with laser ablation than by electrochemical, photochemical or reactive ion etching (RIE). We were able to verify the feasibility of this process by fabrication of micromechanical membrane structures also in bulk 3C-SiC, borosilicate glass, sapphire and Al2O3 ceramic substrates by femtosecond laser (520nm) ablation. On a 350μm thick 4H-SiC substrate we produced an array of 275μm deep and 1000μm to 3000μm of diameter blind holes without damaging the 2μm AlN layer at the back side. In addition we investigated ferroelectric thin films as they can be deposited and micro-patterned by a direct UV-lithography method after the ablation process for a specific membrane design. The risk to harm or damage the function of thin films was eliminated by that means. Some defects in the ablated membranes are also affected by the polarisation of the laser light. Ripple structures oriented perpendicular to the laser polarisation promote creation of pin holes which would perforate a thin membrane. We developed an ablation technique strongly inhibiting formation of ripples and pin poles.

  2. Carbon nanotube embedded PVDF membranes: Effect of solvent composition on the structural morphology for membrane distillation

    NASA Astrophysics Data System (ADS)

    Mapunda, Edgar C.; Mamba, Bhekie B.; Msagati, Titus A. M.

    2017-08-01

    Rapid population increase, growth in industrial and agricultural sectors and global climate change have added significant pressure on conventional freshwater resources. Tapping freshwater from non-conventional water sources such as desalination and wastewater recycling is considered as sustainable alternative to the fundamental challenges of water scarcity. However, affordable and sustainable technologies need to be applied for the communities to benefit from the treatment of non-conventional water source. Membrane distillation is a potential desalination technology which can be used sustainably for this purpose. In this work multi-walled carbon nanotube embedded polyvinylidene fluoride membranes for application in membrane distillation desalination were prepared via non-solvent induced phase separation method. The casting solution was prepared using mixed solvents (N, N-dimethylacetamide and triethyl phosphate) at varying ratios to study the effect of solvent composition on membrane morphological structures. Membrane morphological features were studied using a number of techniques including scanning electron microscope, atomic force microscope, SAXSpace tensile strength analysis, membrane thickness, porosity and contact angle measurements. It was revealed that membrane hydrophobicity, thickness, tensile strength and surface roughness were increasing as the composition of N, N-dimethylacetamide in the solvent was increasing with maximum values obtained between 40 and 60% N, N-dimethylacetamide. Internal morphological structures were changing from cellular structures to short finger-like and sponge-like pores and finally to large macro void type of pores when the amount of N, N-dimethylacetamide in the solvent was changed from low to high respectively. Multi-walled carbon nanotube embedded polyvinylidene fluoride membranes of desired morphological structures and physical properties can be synthesized by regulating the composition of solvents used to prepare the

  3. Developmentally Regulated Sphingolipid Synthesis in African Trypanosomes

    PubMed Central

    Sutterwala, Shaheen S.; Hsu, Fong Fu; Sevova, Elitza S.; Schwartz, Kevin J.; Zhang, Kai; Key, Phillip; Turk, John; Beverley, Stephen M.; Bangs, James D.

    2008-01-01

    Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labeling confirmed stage specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased >3-fold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian SM synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites. PMID:18699867

  4. Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.

    PubMed

    Amado, S; Simões, M J; Armada da Silva, P A S; Luís, A L; Shirosaki, Y; Lopes, M A; Santos, J D; Fregnan, F; Gambarotta, G; Raimondo, S; Fornaro, M; Veloso, A P; Varejão, A S P; Maurício, A C; Geuna, S

    2008-11-01

    Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

  5. Molecular insight into the electrostatic membrane surface potential by 14n/31p MAS NMR spectroscopy: nociceptin-lipid association.

    PubMed

    Lindström, Fredrick; Williamson, Philip T F; Gröbner, Gerhard

    2005-05-11

    Exploiting naturally abundant (14)N and (31)P nuclei by high-resolution MAS NMR (magic angle spinning nuclear magnetic resonance) provides a molecular view of the electrostatic potential present at the surface of biological model membranes, the electrostatic charge distribution across the membrane interface, and changes that occur upon peptide association. The spectral resolution in (31)P and (14)N MAS NMR spectra is sufficient to probe directly the negatively charged phosphate and positively charged choline segment of the electrostatic P(-)-O-CH(2)-CH(2)-N(+)(CH(3))(3) headgroup dipole of zwitterionic DMPC (dimyristoylphosphatidylcholine) in mixed-lipid systems. The isotropic shifts report on the size of the potential existing at the phosphate and ammonium group within the lipid headgroup while the chemical shielding anisotropy ((31)P) and anisotropic quadrupolar interaction ((14)N) characterize changes in headgroup orientation in response to surface potential. The (31)P/(14)N isotropic chemical shifts for DMPC show opposing systematic changes in response to changing membrane potential, reflecting the size of the electrostatic potential at opposing ends of the P(-)-N(+) dipole. The orientational response of the DMPC lipid headgroup to electrostatic surface variations is visible in the anisotropic features of (14)N and (31)P NMR spectra. These features are analyzed in terms of a modified "molecular voltmeter" model, with changes in dynamic averaging reflecting the tilt of the C(beta)-N(+)(CH)(3) choline and PO(4)(-) segment. These properties have been exploited to characterize the changes in surface potential upon the binding of nociceptin to negatively charged membranes, a process assumed to proceed its agonistic binding to its opoid G-protein coupled receptor.

  6. [Advance in research on regulatory mechanism and functions of neutral sphingomyelinse 2].

    PubMed

    Zhang, Lan; Guo, Jun

    2013-10-01

    Neutral sphingomyelinase 2 (nSMase2), which located mainly on the plasma membrane, hydrolyzes sphingomyelin into ceramide and plays an important role in the physiological and pathological regulation of cell apoptosis, cell growth arrest, and inflammation. nSMase2 is also involved in the development of Alzheimer's disease and the bone growth.Under neutral pH and the presence of Ca(2+), Mg(2+), and Mn(+), the activity of nSMase2 is induced by oxidative stress through phosphorylation. Furthermore, the induced interaction of anionic phospholipids and the signaling molecules like receptor for activated C-kinase 1/embryonic ectodermal development with nSMase2 are also crucial mechanisms of protein activation. In the review, recent research advances in the structure and function of nSMase2 and its underlying mechanisms are summarized.

  7. Mixed Matrix Membranes for O2/N2 Separation: The Influence of Temperature

    PubMed Central

    Fernández-Barquín, Ana; Casado-Coterillo, Clara; Valencia, Susana; Irabien, Angel

    2016-01-01

    In this work, mixed matrix membranes (MMMs) composed of small-pore zeolites with various topologies (CHA (Si/Al = 5), LTA (Si/Al = 1 and 5), and Rho (Si/Al = 5)) as dispersed phase, and the hugely permeable poly(1-trimethylsilyl-1-propyne) (PTMSP) as continuous phase, have been synthesized via solution casting, in order to obtain membranes that could be attractive for oxygen-enriched air production. The O2/N2 gas separation performance of the MMMs has been analyzed in terms of permeability, diffusivity, and solubility in the temperature range of 298–333 K. The higher the temperature of the oxygen-enriched stream, the lower the energy required for the combustion process. The effect of temperature on the gas permeability, diffusivity, and solubility of these MMMs is described in terms of the Arrhenius and Van’t Hoff relationships with acceptable accuracy. Moreover, the O2/N2 permselectivity of the MMMs increases with temperature, the O2/N2 selectivities being considerably higher than those of the pure PTMSP. In consequence, most of the MMMs prepared in this work exceeded the Robeson’s upper bound for the O2/N2 gas pair in the temperature range under study, with not much decrease in the O2 permeabilities, reaching O2/N2 selectivities of up to 8.43 and O2 permeabilities up to 4,800 Barrer at 333 K. PMID:27196937

  8. Effect of externally applied electrostatic fields on the surface topography of ceramide-enriched domains in mixed monolayers with sphingomyelin.

    PubMed

    Wilke, Natalia; Maggio, Bruno

    2006-06-20

    Lipid and protein molecules anisotropically oriented at a hydrocarbon-aqueous interface configure a dynamic array of self-organized molecular dipoles. Electrostatic fields applied to lipid monolayers have been shown to induce in-plane migration of domains or phase separation in a homogeneous system. In this work, we have investigated the effect of externally applied electrostatic fields on the distribution of the condensed ceramide-enriched domains in mixed monolayers with sphingomyelin. In these monolayers, the lipids segregate in different phases at all pressures. This allows analyzing by epifluorescence microscopy the effect of the electrostatic field at all lateral pressure because coexistence of lipid domains in condensed state are always present. Our observations indicate that a positive potential applied to an electrode placed over the monolayer promotes a repulsion of the ceramide-enriched domains which is rather insensitive to the film composition, depends inversely on the lateral pressure and exhibits threshold dependence on the in-plane elasticity.

  9. The composition of West Nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis.

    PubMed

    Martín-Acebes, Miguel A; Merino-Ramos, Teresa; Blázquez, Ana-Belén; Casas, Josefina; Escribano-Romero, Estela; Sobrino, Francisco; Saiz, Juan-Carlos

    2014-10-01

    West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV. Importance: West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed for

  10. The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis

    PubMed Central

    Martín-Acebes, Miguel A.; Merino-Ramos, Teresa; Blázquez, Ana-Belén; Casas, Josefina; Escribano-Romero, Estela

    2014-01-01

    ABSTRACT West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV. IMPORTANCE West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed

  11. Ultrathin Composite Polymeric Membranes for CO2 /N2 Separation with Minimum Thickness and High CO2 Permeance.

    PubMed

    Benito, Javier; Sánchez-Laínez, Javier; Zornoza, Beatriz; Martín, Santiago; Carta, Mariolino; Malpass-Evans, Richard; Téllez, Carlos; McKeown, Neil B; Coronas, Joaquín; Gascón, Ignacio

    2017-10-23

    The use of ultrathin films as selective layers in composite membranes offers significant advantages in gas separation for increasing productivity while reducing the membrane size and energy costs. In this contribution, composite membranes have been obtained by the successive deposition of approximately 1 nm thick monolayers of a polymer of intrinsic microporosity (PIM) on top of dense membranes of the ultra-permeable poly[1-(trimethylsilyl)-1-propyne] (PTMSP). The ultrathin PIM films (30 nm in thickness) demonstrate CO 2 permeance up to seven times higher than dense PIM membranes using only 0.04 % of the mass of PIM without a significant decrease in CO 2 /N 2 selectivity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Scanning electron microscopy study of neutrophil membrane tubulovesicular extensions (cytonemes) and their role in anchoring, aggregation and phagocytosis. The effect of nitric oxide

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

    Galkina, Svetlana I.; Molotkovsky, Julian G.; Ullrich, Volker

    2005-04-01

    We have shown that human neutrophils develop dynamic thin and very long tubulovesicular extensions (cytonemes) upon adhesion to fibronectin, if cell spreading was blocked by Na{sup +}-free medium or by 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and cytochalasin D (S. I. Galkina, G. F. Sud'ina and V. Ullrich (2001). Exp. Cell Res. 266, 222-228). In the present work we found that similar in size and behavior tubulovesicular extensions were formed on the neutrophil cell bodies upon adhesion to fibronectin-coated substrata in the presence of the nitric oxide donor diethylamine NONOate. In the presence of the nitric oxide synthase inhibitor N-{omega}-nitro-L-arginine methyl ester,more » neutrophils were well spread and had no microextensions. Using scanning electron microscopy, we demonstrated that tubulovesicular extensions of neutrophils executed long-range adhesion and binding objects for phagocytosis, such as serum-opsonized zymosan particles and erythrocytes. Tubulovesicular extensions anchored neutrophils to substrata in a {beta}1 and {beta}2 integrin-independent, but L-selectin-dependent manner. BODIPY-sphingomyelin impaired development of tubulovesicular extension, and heparitinase 1 played a role in their destruction. Membrane tubulovesicular extensions are supposed to represent protrusions of an intracellular exocytotic traffic and serve as cellular sensory and adhesive organelles. Nitric oxide seems to play a role in regulation of tubulovesicular extensions formation, thus affecting neutrophil adhesive interactions and phagocytosis.« less

  13. Construction of Hierarchical Fouling Resistance Surfaces onto Poly(vinylidene fluoride) Membranes for Combating Membrane Biofouling.

    PubMed

    Li, Xue; Hu, Xuefeng; Cai, Tao

    2017-05-09

    Owing to the highly hydrophobic nature, fluoropolymer membranes usually suffer from serious fouling problem, and therefore largely limited their practical applications. Also, the development of environmentally benign and nonreleasing antifouling coatings onto the inert fluoropolymer membranes remains a great challenge and is of prime importance for various scientific interests and industrial applications. In the present work, a facile and effective approach for the construction of hierarchical fouling resistance surfaces onto the poly(vinylidene fluoride) (PVDF) membranes was developed. Graft copolymers of PVDF with poly(hyperbranched polyglycerol methacrylamide) side chains (PVDF-g-PHPGMA copolymers) were synthesized via reversible addition-fragmentation chain transfer (RAFT) graft copolymerization of pentafluorophenyl methacrylate (PFMA) with the ozone-preactivated PVDF, followed by activated ester-amine reaction of PPFMA chains with amino-terminated hyperbranched polyglycerol (HPG-NH 2 ). The copolymers could be simply processed into microfiltration (MF) membranes with surface-tethered PHPGMA side chains on the membrane and pore surfaces by nonsolvent induced phase inversion. Furthermore, the PVDF-g-PHPGMA-g-PSBMA membrane was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) of zwitterionic monomer, N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SBMA) from the PVDF-g-PHPGMA membrane and pore surfaces. Arise from a synergistic effect of the dendritic architecture of PHPGMA branches and "superhydrophilic" nature of PSBMA brushes, the PVDF-g-PHPGMA-g-PSBMA membranes exhibit superior resistance to protein and bacteria adhesion with insignificant cytotoxicity effects, making the membranes potentially useful for water treatment and biomedical applications. One may find the present study a general and effective method for the fabrication of antifouling fluoropolymer membranes in a controllable and green manner.

  14. Insight into the Putative Specific Interactions between Cholesterol, Sphingomyelin, and Palmitoyl-Oleoyl Phosphatidylcholine

    PubMed Central

    Aittoniemi, Jussi; Niemelä, Perttu S.; Hyvönen, Marja T.; Karttunen, Mikko; Vattulainen, Ilpo

    2007-01-01

    The effects of cholesterol (Chol) on phospholipid bilayers include ordering of the fatty acyl chains, condensing of the lipids in the bilayer plane, and promotion of the liquid-ordered phase. These effects depend on the type of phospholipids in the bilayer and are determined by the nature of the underlying molecular interactions. As for Chol, it has been shown to interact more favorably with sphingomyelin than with most phosphatidylcholines, which in given circumstances leads to formation of lateral domains. However, the exact origin and nature of Chol-phospholipid interactions have recently been subjects of speculation. We examine interactions between Chol, palmitoylsphingomyelin (PSM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in hydrated lipid bilayers by extensive atom-scale molecular dynamics simulations. We employ a tailored lipid configuration: Individual PSM and Chol monomers, as well as PSM-Chol dimers, are embedded in a POPC lipid bilayer in the liquid crystalline phase. Such a setup allows direct comparison of dimeric and monomeric PSMs and Chol, which ultimately shows how the small differences in PSM and POPC structure can lead to profoundly different interactions with Chol. Our analysis shows that direct hydrogen bonding between PSM and Chol does not provide an adequate explanation for their putative specific interaction. Rather, a combination of charge-pairing, hydrophobic, and van der Waals interactions leads to a lower tilt in PSM neighboring Chol than in Chol with only POPC neighbors. This implies improved Chol-induced ordering of PSM's chains over POPC's chains. These findings are discussed in the context of the hydrophobic mismatch concept suggested recently. PMID:17114220

  15. Study of permeability characteristics of membranes

    NASA Technical Reports Server (NTRS)

    Spiegler, K. S.; Moore, R. J.; Leibovitz, J.; Messalem, R. M.

    1972-01-01

    A method is reported for evaluating transport experiments with membranes which is based on conservative fluxes, i.e. fluxes of quantities which do not vary across the membrane in the steady state. Conductance coefficients were calculated for the system: 0.05 N NaCl - C-103 cation-exchange membrane- 0.1 N NaCl. It is concluded that this method can be used to characterize any system of the type - solution-membrane-solution.

  16. Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells.

    PubMed

    Dennison, Sarah Rachel; Harris, Frederick; Brandenburg, Klaus; Phoenix, David Andrew

    2007-11-01

    The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

  17. Simulations of simple Bovine and Homo sapiens outer cortex ocular lens membrane models with a majority concentration of cholesterol.

    PubMed

    Adams, Mark; Wang, Eric; Zhuang, Xiaohong; Klauda, Jeffery B

    2017-11-21

    The lipid composition of bovine and human ocular lens membranes has been probed, and a variety of lipids have been found including phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol (CHOL) with cholesterol being present in particularly high concentrations. In this study, we use the all-atom CHARMM36 force field to simulate binary, ternary, and quaternary mixtures as models of the ocular lens. High concentration of cholesterol, in combination with different and varying diversity of phospholipids (PL) and sphingolipids (SL), affect the structure of the ocular lens lipid bilayer. The following analyses were done for each simulation: surface area per lipid, component surface area per lipid, deuterium order parameters (S CD ), electron density profiles (EDP), membrane thickness, hydrogen bonding, radial distribution functions, clustering, and sterol tilt angle distribution. The S CD show significant bilayer alignment and packing in cholesterol-rich bilayers. The EDP show the transition from liquid crystalline to liquid ordered with the addition of cholesterol. Hydrogen bonds in our systems show the tendency for intramolecular interactions between cholesterol and fully saturated lipid tails for less complex bilayers. But with an increased number of components in the bilayer, the acyl chain of the lipids becomes a less important characteristic, and the headgroup of the lipid becomes more significant. Overall, cholesterol is the driving force of membrane structure of the ocular lens membrane where interactions between cholesterol, PL, and SL determine structure and function of the biomembrane. The goal of this work is to develop a baseline for further study of more physiologically realistic ocular lens lipid membranes. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

    PubMed Central

    Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-01-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

  19. Effect of myristoylated N-terminus of Arf1 on the bending rigidity of phospholipid membranes

    NASA Astrophysics Data System (ADS)

    Burrola Gabilondo, Beatriz; Zhou, Hernan; Randazzo, Paul A.; Losert, Wolfgang

    2010-03-01

    The protein Arf1 is part of the COPI vesicle transport process from the Golgi to the ER. It binds to membranes via a myristoylated N-terminus and it has been shown to tubulate Large Unilamellar Vesicles. The effect of the N-terminus of Arf1 on physical properties of membranes has not been studied, with the exception of curvature. We previously found that the myristoylated N-terminus increases the packing of the lipid molecules, but has no effect on the lateral mobility. We tested the hypothesis that myristoylated peptides affect the bending rigidity of phospholipid Giant Unilamellar Vesicles (GUV). We use optical tweezers to pull tethers from GUV and measure the force of pulling the tether, as well as the retraction speed of the tether once it is released. We also used flicker spectroscopy to estimate the values of the mechanical properties of GUV. We will present results of the force and tether retraction measurements, as well as mechanical properties estimates from flicker, for GUV in the presence of varying concentrations of myristoylated and non-myristoylated N-terminus of Arf1, and compare these with measurements for GUV in the absence of peptide.

  20. Targeting N-acyl-homoserine-lactones to mitigate membrane biofouling based on quorum sensing using a biofouling reducer.

    PubMed

    Siddiqui, Muhammad Faisal; Sakinah, Mimi; Singh, Lakhveer; Zularisam, A W

    2012-10-31

    Exploring novel biological anti-quorum sensing (QS) agents to control membrane biofouling is of great worth in order to allow sustainable performance of membrane bioreactors (MBRs) for wastewater treatment. In recent studies, QS inhibitors have provided evidence of alternative route to control membrane biofouling. This study investigated the role of Piper betle extract (PBE) as an anti-QS agent to mitigate membrane biofouling. Results demonstrated the occurrence of the N-acyl-homoserine-lactone (AHL) autoinducers (AIs), correlate QS activity and membrane biofouling mitigation. The AIs production in bioreactor was confirmed using an indicator strain Agrobacterium tumefaciens (NTL4) harboring plasmid pZLR4. Moreover, three different AHLs were found in biocake using thin layer chromatographic analysis. An increase in extracellular polymeric substances (EPS) and transmembrane pressure (TMP) was observed with AHL activity of the biocake during continuous MBR operation, which shows that membrane biofouling was in close relationship with QS activity. PBE was verified to mitigate membrane biofouling via inhibiting AIs production. SEM analysis further confirmed the effect of PBE on EPS and biofilm formation. These results exhibited that PBE could be a novel agent to target AIs for mitigation of membrane biofouling. Further work can be carried out to purify the active compound of Piper betle extract to target the QS to mitigate membrane biofouling. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Consequences of ions and pH on the supramolecular organization of sphingomyelin and sphingomyelin/cholesterol bilayers.

    PubMed

    Chemin, Caroline; Bourgaux, Claudie; Péan, Jean-Manuel; Pabst, Georg; Wüthrich, Patrick; Couvreur, Patrick; Ollivon, Michel

    2008-06-01

    For drug delivery purpose the anticancer drug S12363 was loaded into ESM/Chol-liposomes using either a pH or an ammonium gradient. Association between the drug and the liposome depends markedly on the liposome membrane structure. Thus, ESM and ESM/Chol bilayer organization had been characterized by coupled DSC and XRDT as a function of both cholesterol concentration and aqueous medium composition. ESM bilayers exhibited a ripple lamellar gel phase P(beta') below the melting temperature and adopted a L(beta)-like gel phase upon Chol insertion. Supramolecular organization of ESM and ESM/Chol bilayers was not modified by citrate buffer or ammonium sulfate solution whatever the pH (3< or = pH < or =7). Nevertheless, in ESM bilayer, ammonium sulfate salt induced a peculiar organization of head groups, leading to irregular d-spacing and weakly correlated bilayers. Moreover, in the presence of salts, a weakening of van der Waals attraction forces was seen and led to a swelling of the water layer.

  2. Dietary Milk Sphingomyelin Prevents Disruption of Skin Barrier Function in Hairless Mice after UV-B Irradiation.

    PubMed

    Oba, Chisato; Morifuji, Masashi; Ichikawa, Satomi; Ito, Kyoko; Kawahata, Keiko; Yamaji, Taketo; Asami, Yukio; Itou, Hiroyuki; Sugawara, Tatsuya

    2015-01-01

    Exposure to ultraviolet-B (UV-B) irradiation causes skin barrier defects. Based on earlier findings that milk phospholipids containing high amounts of sphingomyelin (SM) improved the water content of the stratum corneum (SC) in normal mice, here we investigated the effects of dietary milk SM on skin barrier defects induced by a single dose of UV-B irradiation in hairless mice. Nine week old hairless mice were orally administrated SM (146 mg/kg BW/day) for a total of ten days. After seven days of SM administration, the dorsal skin was exposed to a single dose of UV-B (20 mJ/cm2). Administration of SM significantly suppressed an increase in transepidermal water loss and a decrease in SC water content induced by UV-B irradiation. SM supplementation significantly maintained covalently-bound ω-hydroxy ceramide levels and down-regulated mRNA levels of acute inflammation-associated genes, including thymic stromal lymphopoietin, interleukin-1 beta, and interleukin-6. Furthermore, significantly higher levels of loricrin and transglutaminase-3 mRNA were observed in the SM group. Our study shows for the first time that dietary SM modulates epidermal structures, and can help prevent disruption of skin barrier function after UV-B irradiation.

  3. Lithography-based fabrication of nanopore arrays in freestanding SiN and graphene membranes

    NASA Astrophysics Data System (ADS)

    Verschueren, Daniel V.; Yang, Wayne; Dekker, Cees

    2018-04-01

    We report a simple and scalable technique for the fabrication of nanopore arrays on freestanding SiN and graphene membranes based on electron-beam lithography and reactive ion etching. By controlling the dose of the single-shot electron-beam exposure, circular nanopores of any size down to 16 nm in diameter can be fabricated in both materials at high accuracy and precision. We demonstrate the sensing capabilities of these nanopores by translocating dsDNA through pores fabricated using this method, and find signal-to-noise characteristics on par with transmission-electron-microscope-drilled nanopores. This versatile lithography-based approach allows for the high-throughput manufacturing of nanopores and can in principle be used on any substrate, in particular membranes made out of transferable two-dimensional materials.

  4. Four Novel p.N385K, p.V36A, c.1033–1034insT and c.1417–1418delCT Mutations in the Sphingomyelin Phosphodiesterase 1 (SMPD1) Gene in Patients with Types A and B Niemann-Pick Disease (NPD)

    PubMed Central

    Manshadi, Masoumeh Dehghan; Kamalidehghan, Behnam; Keshavarzi, Fatemeh; Aryani, Omid; Dadgar, Sepideh; Arastehkani, Ahoora; Tondar, Mahdi; Ahmadipour, Fatemeh; Meng, Goh Yong; Houshmand, Massoud

    2015-01-01

    Background: Types A and B Niemann-Pick disease (NPD) are autosomal-recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene. Methods: In order to determine the prevalence and distribution of SMPD1 gene mutations, the genomic DNA of 15 unrelated Iranian patients with types A and B NPD was examined using PCR, DNA sequencing and bioinformatics analysis. Results: Of 8 patients with the p.G508R mutation, 5 patients were homozygous, while the other 3 were heterozygous. One patient was heterozygous for both the p.N385K and p.G508R mutations. Another patient was heterozygous for both the p.A487V and p.G508R mutations. Two patients (one homozygous and one heterozygous) showed the p.V36A mutation. One patient was homozygous for the c.1033–1034insT mutation. One patient was homozygous for the c.573delT mutation, and 1 patient was homozygous for the c.1417–1418delCT mutation. Additionally, bioinformatics analysis indicated that two new p.V36A and p.N385K mutations decreased the acid sphingomyelinase (ASM) protein stability, which might be evidence to suggest the pathogenicity of these mutations. Conclusion: with detection of these new mutations, the genotypic spectrum of types A and B NPD is extended, facilitating the definition of disease-related mutations. However, more research is essential to confirm the pathogenic effect of these mutations. PMID:25811928

  5. Ultem((R))/ZIF-8 mixed matrix hollow fiber membranes for CO2/N-2 separations

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

    Dai, Y; Johnson, JR; Karvan, O

    2012-05-15

    Organic-inorganic hybrid (mixed matrix) membranes can potentially extend the separation performance of traditional polymeric materials while maintaining processing convenience. Although many dense films studies have been reported, there have been few reported cases of these materials being successfully extended to asymmetric hollow fibers. In this work we report the first successful production of mixed matrix asymmetric hollow fiber membranes containing metal-organic-framework (MOF) ZIF-8 fillers. Specifically, we have incorporated ZIF-8 into a polyetherimide (Ultem((R)) 1000) matrix and produced dual-layer asymmetric hollow fiber membranes via the dry jet-wet quench method. The outer separating layer of these composite fibers contains 13 wt% (17more » vol%) of ZIF-8 filler. These membranes have been tested over a range of temperatures and pressures for a variety of gas pairs. An increase in separation performance for the CO2/N-2 gas pairs was observed for both pure gas and mixed gas feeds. (C) 2012 Elsevier B.V. All rights reserved.« less

  6. The membrane biofilm reactor: the natural partnership of membranes and biofilm.

    PubMed

    Rittmann, B E

    2006-01-01

    Many exciting new technologies for water-quality control combine microbiological processes with adsorption, advanced oxidation, a membrane or an electrode to improve performance, address emerging contaminants or capture renewable energy. An excellent example is the H2-based membrane biofilm reactor (MBfR), which delivers H2 gas to a biofilm that naturally accumulates on the outer surface of a bubbleless membrane. Autotrophic bacteria in the biofilm oxidise the H2 and use the electrons to reduce NO3-, CIO4- and other oxidised contaminants. This natural partnership of membranes and biofilm makes it possible to gain many cost, performance and simplicity advantages from using H2 as the electron donor for microbially catalysed reductions. The MBfR has been demonstrated for denitrification in drinking water; reduction of perchlorate in groundwater; reduction of selenate, chromate, trichloroethene and other emerging contaminants; advanced N removal in wastewater treatment and autotrophic total-N removal.

  7. Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers.

    PubMed

    Mote, Kaustubh R; Gopinath, T; Traaseth, Nathaniel J; Kitchen, Jason; Gor'kov, Peter L; Brey, William W; Veglia, Gianluigi

    2011-11-01

    Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring (1)H-(15)N dipolar couplings (DC) and (15)N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles' heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([(1)H,(15)N]-SE-PISEMA-PDSD). The incorporation of 2D (15)N/(15)N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the (15)N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers. © Springer Science+Business Media B.V. 2011

  8. Physically Gelled Room-Temperature Ionic Liquid-Based Composite Membranes for CO2/N-2 Separation: Effect of Composition and Thickness on Membrane Properties and Performance

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

    Nguyen, PT; Voss, BA; Wiesenauer, EF

    2013-07-03

    An aspartame-based, low molecular-weight organic gelator (LMOG) was used to form melt-infused and composite membranes with two different imidazolium-based room-temperature ionic liquids (RTILs) for CO2 separation from N-2. Previous work demonstrated that LMOGs can gel RTILs at low, loading levels, and this aspartame-based LMOG was selected because it has been reported to gel a large number of RTILs. The imidazolium-based RTILs were used because of their inherent good properties for CO2/light gas separations. Analysis of the resulting bulk RTIL/LMOG physical gels showed that these materials have high sol-gel transition temperatures (ca. 135 degrees C) suitable for flue gas applications. Gasmore » permeabilities and burst pressure measurements of thick, melt infused membranes revealed a trade-off between high CO2 permeabilities and good mechanical stability as a function of the LMOG loading. Defect-free, composite membranes of the gelled RTILs were successfully fabricated by choosing an appropriate porous membrane support (hydrophobic PTFE) using a suitable coating technique (roller coating). The thicknesses of the applied composite gel layers ranged from 10.3 to 20.7 mu m, which represents an order of magnitude decrease in active layer thickness, compared to the original melt-infused gel RTIL membranes.« less

  9. Improvement in hemocompatibility of chitosan/soy protein composite membranes by heparinization.

    PubMed

    Wang, Xiaomei; Shi, Na; Chen, Yan; Li, Chen; Du, Xinshen; Jin, Weihua; Chen, Yun; Chang, Peter R

    2012-01-01

    To improve the hemocompatibility of chitosan/soy protein isolate composite membranes by heparinization. Chitosan/soy protein isolate membranes (ChS-n, n=0, 10 and 30, corresponding to the soy protein isolate content in the membranes) and heparinized ChS-n membranes (HChS-n) were prepared by blending in dilute HAc/NaAc solution. The hemocompatibility of ChS-n and HChS-n membranes were comparatively evaluated by measuring surface heparin density, blood platelet adhesion, plasma recalcification time (PRT), thrombus formation and hemolysis assay. The surface heparin density analysis showed that heparinized chitosan/SPI soy protein isolate membranes have been successfully prepared by blending. The density of heparin on the surface of HChS-n membranes was in the range of 0.67-1.29 μg/cm2. The results of platelet adhesion measurement showed that the platelet adhesion numbers of HChS-n membranes were lower than those of the corresponding ChS-n membranes. The PRT of the HChS-0, HChS-10 and HChS-30 membranes were around 292, 306 and 295 s, respectively, which were longer than the corresponding ChS-0 (152 s), ChS-10 (204 s) and ChS-30 (273 s) membranes. The hemolysis rate of HChS-n membranes was lower than 1%. The hemocompatibility of ChS membranes could be improved by blending with heparin. Compared with ChS membranes, HChS membranes showed lower platelet adhesion, longer PRT, higher BCI, significant thromboresistivity and a lower hemolysis rate due to the heparinization. This widens the application of chitosan and soy protein-based biomaterials that may come in contact with blood.

  10. Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells[S

    PubMed Central

    Kouzel, Ivan U.; Pohlentz, Gottfried; Storck, Wiebke; Radamm, Lena; Hoffmann, Petra; Bielaszewska, Martina; Bauwens, Andreas; Cichon, Christoph; Schmidt, M. Alexander; Mormann, Michael; Karch, Helge; Müthing, Johannes

    2013-01-01

    Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin. PMID:23248329

  11. Alteration of the lipid composition of rat testicular plasma membranes by dietary (n-3) fatty acids changes the responsiveness of Leydig cells and testosterone synthesis.

    PubMed

    Sebokova, E; Garg, M L; Wierzbicki, A; Thomson, A B; Clandinin, M T

    1990-06-01

    Experiments were conducted to assess whether changing dietary fat composition altered phospholipid composition of rat testicular plasma membranes in a manner that altered receptor-mediated action of luteinizing hormone (LH)/human chorionic gonadotropin (hCG). Weanling rats were fed diets that provided high or low cholesterol intakes and that were enriched with linseed oil, fish oil or beef tallow for 4 wk. Feeding diets high in (n-3) fatty acids decreased plasma and testicular plasma membrane 20:4(n-6) content. A marked reduction of the 22:5(n-6) content and an increase in the 22:6(n-3) content of testicular plasma membrane was found only in animals fed fish oil. A decrease in binding capacity of the gonadotropin (LH/hCG) receptor in the plasma membrane, with no change in receptor affinity, was observed for animals fed either linseed oil or fish oil diets. Dietary treatments that raised plasma membrane cholesterol content and the cholesterol to phospholipid ratio in the membrane were associated with increased binding capacity of the gonadotropin receptor. Feeding diets high in 18:3(n-3) vs. those high in fish oil altered receptor-mediated adenylate cyclase activity in a manner that depended on the level of dietary cholesterol. Feeding diets high in cholesterol or fish oil increased basal and LH-stimulated testosterone synthesis relative to that in animals fed the low cholesterol diet containing linseed oil. It is concluded that changing the fat composition of the diet alters the phospholipid composition of rat testicular plasma membranes and that this change in composition influences membrane-mediated unmasking of gonadotropin receptor-mediated action in testicular tissue.

  12. Dynamics of a fluctuating semi-flexible membrane

    NASA Astrophysics Data System (ADS)

    Tukdarian, Nathaniel; Huang, Aiqun; Adhikari, Ramesh; Bhattacharya, Aniket

    2015-03-01

    We report our preliminary studies of conformations and dynamics of a fluctuating semi-flexible membrane. Our model of membrane with linear dimension L consists of N2 (L = Nbl) excluded volume beads connected by anharmonic springs. The stiffness of the membrane is controlled by adjusting the strength κb of the bending potential Ubend =κb(1-n̂i .n̂j) between adjacent elementary plaquettes consisting of three beads at the corners connected by bonds and characterized by normal unit vectors n̂i and n̂j. We study the conformations and dynamic fluctuations of the membrane using Brownian dynamics simulation. We show how the radius of gyration scales with N and κb, and study characteristics of the transverse fluctuations, the root-mean-square displacement of the center of mass, and the dynamics of the end monomers at each corner.

  13. Separation of n-hexane/acetone mixtures by pervaporation using high density polyethylene/ethylene propylene diene terpolymer rubber blend membranes.

    PubMed

    Kumar, P V Anil; Anilkumar, S; Varughese, K T; Thomas, Sabu

    2012-01-15

    Polymer membranes were prepared by blending high density polyethylene (HDPE) with ethylene propylene diene terpolymer rubber (EPDM). These blend membranes were evaluated for the selective separation of n-hexane from acetone. The flux and selectivity of the membranes were determined both as a function of the blend composition and feed mixture composition. Results showed that polymer blending method could be very useful to develop new membranes with improved selectivity. Pervaporation properties could be optimized by adjusting the blend composition. The effects of blend ratio, feed composition, and penetrant size on the pervaporation process were analyzed. The permeation properties have been explained on the basis of interaction between the membrane and solvents and blend morphology. Flux increases with increasing alkane content in the feed composition. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

    PubMed Central

    MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

    2015-01-01

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

  15. Phenomenon of hot-cold hemolysis: chelator-induced lysis of sphingomyelinase-treated erythrocytes.

    PubMed Central

    Smyth, C J; Möllby, R; Wadström, T

    1975-01-01

    Staphylococcus aureus produces a phospholipase C specific for sphingomyelin (beta-hemolysin). Erythrocytes with approximately 50% sphingomyelin in their membranes, e.g., from sheep, have been shown to have up to 60% of this phospholipid hydrolyzed by this enzyme at 37 C in isotonic buffered saline without hemolysis. Cooling of sphingomyelinase C-treated erythrocytes to 4 C causes complete lysis of the cells, a phenomenon known as hot-cold hemolysis. The addition of ethylenediaminetetraacetate (EDTA) to sheep erythrocytes preincubated with sphingomyelinase C was found to induce rapid hemolysis at 37 C. The treated cells became susceptible to chelator-induced hemolysis and to hot-cold hemolysis simultaneously, and the degree of lysis of both mechanisms increased equally with prolonged preincubation with sphingomyelinase C. Erythrocytes of species not readily susceptible to hot-cold hemolysis were equally insusceptible to chelator-induced lysis. Chelators of the EDTA series were the most effective, whereas chelators more specific for Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+ were without effect. The rate of chelator-induced lysis was dependent on the preincubation period with beta-hemolysin and on the concentration of chelator added. The optimal concentration of EDTA was found to equal the amount of exogenously added Mg2+, a cation necessary for sphingomyelinase C activity. Hypotonicity increased the rate of chelator-induced hemolysis, whereas increasing the osmotic pressure to twice isotonic completely inhibited chelator-induced lysis. The data suggest that exogenously added and/or membrane-bound divalent cations are important for the stability of sphingomyelin-depleted membranes. The phenomenon of hot-cold hemolysis may be a consequence of the temperature dependence of divalent ion stabilization. Images PMID:333

  16. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    PubMed

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  17. Animal cells dependent on exogenous phosphatidylcholine for membrane biogenesis.

    PubMed Central

    Esko, J D; Nishijima, M; Raetz, C R

    1982-01-01

    A Chinese hamster ovary cell (CHO) mutant (strain 58), defective in CDP-choline synthetase (cholinephosphate cytidylyltransferase; CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15), is temperature sensitive for growth and contains less than half of the normal amount of phosphatidylcholine under nonpermissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc. Natl. Acad. Sci. USA 77, 5192-5196]. We now report that the addition of 40 microM egg phosphatidylcholine or lysophosphatidylcholine to the medium suppresses the temperature sensitivity of mutant 58 and permits the growth of colonies at the restrictive temperature. Phospholipids with different polar headgroups, lipoprotein-bound phospholipids, sphingomyelin, and glycerophosphocholine do not support prolonged growth at 40 degrees C, whereas phosphatidylcholine analogs such as phosphatidyldimethylethanolamine, D-phosphatidylcholine, and beta-phosphatidylcholine are quite effective. A broad range of saturated phosphatidylcholines, especially those with fatty acids 12-18 carbons in length, suppresses the phenotype. Phospholipids containing ether-linked hydrocarbons are ineffective, whereas polyunsaturated phosphatidylcholines are toxic. Residual endogenous synthesis of phosphatidylcholine by the mutant is not stimulated under conditions of phenotypic bypass, but the uptake of exogenous lipid is enhanced considerably compared to the wild type. Our findings demonstrate that exogenous phospholipid can provide at least 50% of the phosphatidylcholine required for membrane biogenesis in animal cells and that uptake of exogenous phospholipids may be regulated. PMID:6281780

  18. Anion permselective membrane

    NASA Technical Reports Server (NTRS)

    Hodgdon, R. B.; Waite, W. A.

    1982-01-01

    The synthesis and fabrication of polymeric anion permselective membranes for redox systems are discussed. Variations of the prime candidate anion membrane formulation to achieve better resistance and/or lower permeability were explored. Processing parameters were evaluated to lower cost and fabricate larger sizes. The processing techniques to produce more membranes per batch were successfully integrated with the fabrication of larger membranes. Membranes of about 107 cm x 51 cm were made in excellent yield. Several measurements were made on the larger sample membranes. Among the data developed were water transport and transference numbers for these prime candidate membranes at 20 C. Other work done on this system included characterization of a number of specimens of candidate membranes which had been returned after service lives of up to sixteen months. Work with new polymer constituents, with new N.P.'s, catalysts and backing fabrics is discussed. Some work was also done to evaluate other proportions of the ingredients of the prime candidate system. The adoption of a flow selectivity test at elevated temperature was explored.

  19. Design of poly(vinylidene fluoride)-g-p(hydroxyethyl methacrylate-co-N-isopropylacrylamide) membrane via surface modification for enhanced fouling resistance and release property

    NASA Astrophysics Data System (ADS)

    Zhao, Guili; Chen, Wei Ning

    2017-03-01

    Thermo-sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm), hydrophilic polymer poly(hydroxyethyl methacrylate) (PHEMA) and copolymer p(hydroxyethyl methacrylate-co-N-isopropylacrylamide) [P(HEMA-co-NIPAAm)] were synthesized onto poly(vinylidene fluoride) (PVDF) membrane via atom transfer radical polymerization (ATRP) in order to improve not only fouling resistance but also fouling release property. The physicochemical properties of membranes including hydrophilicity, morphology and roughness were examined by contact angle analyzer, scanning electron microscopy (SEM), and atomic force microscopy (AFM), respectively. The antifouling property of membranes was improved remarkably after surface modification according to protein and bacterial adhesion testing, and filtration experiment. Minimum protein adsorption and bacterial adhesion were both obtained on PVDF-g-P(HEMA-co-NIPAAm) membrane, with reduction by 44% and 71% respectively compared to the pristine membrane. The minimum bacterial cells after detachment at 25 °C were observed on the PVDF-g-P(HEMA-co-NIPAAm) membrane with the detachment rate of 77%, indicating high fouling release property. The filtration testing indicated that the copolymer modified membrane exhibited high resistance to protein fouling and the foulant on the surface was released and removed easily by washing, suggesting high fouling release and easy-cleaning capacity. This study provides useful insight in the combined "fouling resistance" and "fouling release" property of P(HEMA-co-NIPAAm) for PVDF membrane modification, even for other types of the membrane in wide application.

  20. Functional role of the extracellular N-terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization.

    PubMed

    Lindner, Diana; Walther, Cornelia; Tennemann, Anja; Beck-Sickinger, Annette G

    2009-01-01

    The N terminus is the most variable element in G protein-coupled receptors (GPCRs), ranging from seven residues up to approximately 5900 residues. For family B and C GPCRs it is described that at least part of the ligand binding site is located within the N terminus. Here we investigated the role of the N terminus in the neuropeptide Y receptor family, which belongs to the class A of GPCRs. We cloned differentially truncated Y receptor mutants, in which the N terminus was partially or completely deleted. We found, that eight amino acids are sufficient for full ligand binding and signal transduction activity. Interestingly, we could show that no specific amino acids but rather the extension of the first transmembrane helix by any residues is sufficient for receptor activity but also for membrane integration in case of the hY(1) and the hY(4) receptors. In contrast, the complete deletion of the N terminus in the hY(2) receptors resulted in a mutant that is fully integrated in the membrane but does not bind the ligand very well and internalizes much slower compared to the wild type receptor. Interestingly, also these effects could be reverted by any N-terminal extension. Accordingly, the most important function of the N termini seems to be the stabilization of the first transmembrane helix to ensure the correct receptor structure, which obviously is essential for ligand binding, integration into the cell membrane and receptor internalization.

  1. Tunable water desalination across Graphene Oxide Framework membranes

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

    Nicolai, Adrien; Sumpter, Bobby G; Meunier, V.

    2014-01-01

    The performance of graphene oxide framework (GOF) membranes for water desalination is assessed using classical molecular dynamics (MD) simulations. The coupling between water permeability and salt rejection GOF membranes is studied as a function of linker concentration n, thickness h and applied pressure DP. The simulations reveal that water permeability in GOF-(n,h) membranes can be tuned from 5 (n = 32 and h = 6.5 nm) to 400 L/cm2/day/MPa (n = 64 and h = 2.5 nm) and follows the law Cnh an . For a given pore size (n = 16 or 32), water permeability of GOF membranes increasesmore » when the pore spacing decreases, whereas for a given pore spacing (n = 32 or 64), water permeability increases by up to two orders of magnitude when the pore size increases. Furthermore, for linker concentrations n 32, the high water permeability corresponds to a 100% salt rejection, elevating this type of GOF membrane as an ideal candidate for water desalination. Compared to experimental performance of reverse osmosis membranes, our calculations suggest that under the same conditions of applied pressure and characteristics of membranes (DP 10 MPa and h 100 nm), one can expect a perfect salt rejection coupled to a water permeability two orders of magnitude higher than existing technologies, i.e., from a few cL/cm2/day/MPa to a few L/cm2/day/MPa.« less

  2. Membrane lipid profiles of coral responded to zinc oxide nanoparticle-induced perturbations on the cellular membrane.

    PubMed

    Tang, Chuan-Ho; Lin, Ching-Yu; Lee, Shu-Hui; Wang, Wei-Hsien

    2017-06-01

    Zinc oxide nanoparticles (nZnOs) released from popular sunscreens used during marine recreation apparently endanger corals; however, the known biological effects are very limited. Membrane lipids constitute the basic structural element to create cell a dynamic structure according to the circumstance. Nano-specific effects have been shown to mechanically perturb the physical state of the lipid membrane, and the cells accommodating the actions of nZnOs can be involved in the alteration of the membrane lipid composition. To gain insight into the effects of nanoparticles on coral, glycerophosphocholine (GPC) profiling of the coral Seriatopora caliendrum exposed to nZnOs was performed in this study. Increasing lyso-GPCs, docosapentaenoic acid-possessing GPCs and docosahexaenoic acid-possessing GPCs and decreasing arachidonic acid-possessing GPCs were the predominant changes responded to nZnO exposure in the coral. A backfilling of polyunsaturated plasmanylcholines was observed in the coral exposed to nZnO levels over a threshold. These changes can be logically interpreted as an accommodation to nZnOs-induced mechanical disturbances in the cellular membrane based on the biophysical properties of the lipids. Moreover, the coral demonstrated a difference in the changes in lipid profiles between intra-colonial functionally differentiated polyps, indicating an initial membrane composition-dependent response. Based on the physicochemical properties and physiological functions of these changed lipids, some chronic biological effects can be incubated once the coral receives long-term exposure to nZnOs. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Multichannel detection of ionic currents through two nanopores fabricated on integrated Si3N4 membranes.

    PubMed

    Yanagi, Itaru; Akahori, Rena; Aoki, Mayu; Harada, Kunio; Takeda, Ken-Ichi

    2016-08-16

    Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip.

  4. Does cholesterol preferentially pack in lipid domains with saturated sphingomyelin over phosphatidylcholine? A comprehensive monolayer study combined with grazing incidence X-ray diffraction and Brewster angle microscopy experiments.

    PubMed

    Wydro, Paweł; Flasiński, Michał; Broniatowski, Marcin

    2013-05-01

    In this work, the Langmuir monolayers were used as a model for the analysis of the influence of cholesterol on 1,2-distearoyl-sn-glycero-3-phosphocholine - DSPC and stearoyl sphingomyelin - SSM, as well as their equimolar mixture. The aim of these studies was to compare the affinity of cholesterol to sphingomyelin and phosphatidylcholine and discuss the effectiveness of cholesterol packing with these phospholipids. The experiments involved the registration of the surface pressure-area isotherms combined with the application of Brewster angle microscopy (BAM) images and grazing incidence X-ray diffraction methods. We have performed a thorough analysis of the properties of both one-component DSPC and SSM films as well as their 1:1 mixture. Next, the effect of cholesterol on these systems was verified based on the results for 2:1 SSM/Chol, 2:1 DSPC/Chol, and 1:1:1 DSPC/SSM/Chol mixtures. It was found that both phospholipids form highly condensed monolayers, however, they differ in the orientation of acyl chains, namely the acyl chains are more tilted in DSPC film as compared to SSM monolayer as well as DSPC/SSM mixture. Furthermore, the area contraction provoked by the addition of cholesterol was found to be more pronounced for DSPC monolayer than in DSPC/SSM and SSM films. However, all the collected results allow one to postulate that the ability of cholesterol to form ordered domains with DSPC and SSM is similar and is predominantly driven by hydrophobic forces between molecules. The differences in the area condensation induced by cholesterol on the studied phospholipids films results from differences in molecular organization of pure phospholipids films rather than specific cholesterol-phospholipid interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. A ROIC for Mn(TPP)Cl-DOP-THF-Polyhema PVC membrane modified n-channel Si3N4 ISFET sensitive to histamine.

    PubMed

    Samah, N L M A; Lee, Khuan Y; Sulaiman, S A; Jarmin, R

    2017-07-01

    Intolerance of histamine could lead to scombroid poisoning with fatal consequences. Current detection methods for histamine are wet laboratory techniques which employ expensive equipment that depends on skills of seasoned technicians and produces delayed test analysis result. Previous works from our group has established that ISFETs can be adapted for detecting histamine with the use of a novel membrane. However, work to integrate ISFETs with a readout interfacing circuit (ROIC) circuit to display the histamine concentration has not been reported so far. This paper concerns the development of a ROIC specifically to integrate with a Mn(TPP)Cl-DOP-THF-Polyhema PVC membrane modified n-channel Si3N4 ISFET to display the histamine concentration. It embodies the design of constant voltage constant current (CVCC) circuit, amplification circuit and micro-controller based display circuit. A DC millivolt source is used to substitute the membrane modified ISFET as preliminary work. Input is histamine concentration corresponding to the safety level designated by the Food and Drugs Administration (FDA). Results show the CVCC circuit makes the output follows the input and keeps VDS constant. The amplification circuit amplifies the output from the CVCC circuit to the range 2.406-4.888V to integrate with the microcontroller, which is programmed to classify and display the histamine safety level and its corresponding voltage on a LCD panel. The ROIC could be used to produce direct output voltages corresponding to histamine concentrations, for in-situ applications.

  6. Triton promotes domain formation in lipid raft mixtures.

    PubMed

    Heerklotz, H

    2002-11-01

    Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.

  7. Triton promotes domain formation in lipid raft mixtures.

    PubMed Central

    Heerklotz, H

    2002-01-01

    Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds. PMID:12414701

  8. Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

    PubMed

    MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

    2015-04-24

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Modeling Membrane Deformations and Lipid Demixing upon Protein-Membrane Interaction: The BAR Dimer Adsorption

    PubMed Central

    Khelashvili, George; Harries, Daniel; Weinstein, Harel

    2009-01-01

    We use a self-consistent mean-field theory, designed to investigate membrane reshaping and lipid demixing upon interaction with proteins, to explore BAR domains interacting with large patches of lipid membranes of heterogeneous compositions. The computational model includes contributions to the system free energy from electrostatic interactions and elastic energies of the membrane, as well as salt and lipid mixing entropies. The results from our simulation of a single adsorbing Amphiphysin BAR dimer indicate that it is capable of stabilizing a significantly curved membrane. However, we predict that such deformations will occur only for membrane patches that have the inherent propensity for high curvature, reflected in the tendency to create local distortions that closely match the curvature of the BAR dimer itself. Such favorable preconditioning for BAR-membrane interaction may be the result of perturbations such as local lipid demixing induced by the interaction, or of a prior insertion of the BAR domain's amphiphatic N-helix. From our simulations it appears that local segregation of charged lipids under the influence of the BAR dimer cannot produce high enough asymmetry between bilayer leaflets to induce significant bending. In the absence of additional energy contributions that favor membrane asymmetry, the membrane will remain nearly flat upon single BAR dimer adsorption, relative to the undulation expected from thermal fluctuations. Thus, we conclude that the N-helix insertions have a critical mechanistic role in the local perturbation and curving of the membrane, which is then stabilized by the electrostatic interaction with the BAR dimer. We discuss how these results can be used to estimate the tendency of BARs to bend membranes in terms of a spatially nonisotropic spontaneous curvature. PMID:19751667

  10. Separation of copper ions from iron ions using PVA-g-(acrylic acid/N-vinyl imidazole) membranes prepared by radiation-induced grafting.

    PubMed

    Ajji, Zaki; Ali, Ali M

    2010-01-15

    Acrylic acid (AAc), N-vinyl imidazole (Azol) and their binary mixtures were graft copolymerized onto poly(vinyl alcohol) membranes using gamma irradiation. The ability of the grafted membranes to separate Cu ions from Fe ions was investigated with respect to the grafting yield and the pH of the feed solution. The data showed that the diffusion of copper ions from the feed compartment to the receiver compartment depends on the grafting yield of the membranes and the pH of the feed solution. To the contrary, iron ions did not diffuse through the membranes of all grafting yields. However, a limited amount of iron ions diffused in strong acidic medium. This study shows that the prepared membranes could be considered for the separation of copper ions from iron ions. The temperature of thermal decomposition of pure PVA-g-AAc/Azol membrane, PVA-g-AAc/Azol membrane containing copper ions, and PVA-g-AAc/Azol membrane containing iron ions were determined using TGA analyzer. It was shown that the presence of Cu and Fe ions increases the decomposition temperature, and the membranes bonded with iron ions are more stable than those containing copper ions.

  11. The Probability of Neonatal Respiratory Distress Syndrome as a Function of Gestational Age and Lecithin/Sphingomyelin Ratio

    PubMed Central

    St. Clair, Caryn; Norwitz, Errol R.; Woensdregt, Karlijn; Cackovic, Michael; Shaw, Julia A.; Malkus, Herbert; Ehrenkranz, Richard A.; Illuzzi, Jessica L.

    2011-01-01

    We sought to define the risk of neonatal respiratory distress syndrome (RDS) as a function of both lecithin/sphingomyelin (L/S) ratio and gestational age. Amniotic fluid L/S ratio data were collected from consecutive women undergoing amniocentesis for fetal lung maturity at Yale-New Haven Hospital from January 1998 to December 2004. Women were included in the study if they delivered a live-born, singleton, nonanomalous infant within 72 hours of amniocentesis. The probability of RDS was modeled using multivariate logistic regression with L/S ratio and gestational age as predictors. A total of 210 mother-neonate pairs (8 RDS, 202 non-RDS) met criteria for analysis. Both gestational age and L/S ratio were independent predictors of RDS. A probability of RDS of 3% or less was noted at an L/S ratio cutoff of ≥3.4 at 34 weeks, ≥2.6 at 36 weeks, ≥1.6 at 38 weeks, and ≥1.2 at term. Under 34 weeks of gestation, the prevalence of RDS was so high that a probability of 3% or less was not observed by this model. These data describe a means of stratifying the probability of neonatal RDS using both gestational age and the L/S ratio and may aid in clinical decision making concerning the timing of delivery. PMID:18773379

  12. Influence of nanoparticles on filterability of fruit-juice industry wastewater using submerged membrane bioreactor.

    PubMed

    Demirkol, Guler Turkoglu; Dizge, Nadir; Acar, Turkan Ormanci; Salmanli, Oyku Mutlu; Tufekci, Nese

    2017-07-01

    In this study, polyethersulfone (PES) ultrafiltration membrane surface was modified with nano-sized zinc oxide (nZnO) and silver (nAg) to improve the membrane filterability of the mixed liquor and used to treat fruit-juice industry wastewater in a submerged membrane bioreactor (MBR). The nAg was synthesized using three different methods. In the first method, named as nAg-M1, PES membrane was placed on the membrane module and nAg solution was passed through the membrane for 24 h at 25 ± 1 °C. In the second method, named as nAg-M2, PES membrane was placed in a glass container and it was shaken for 24 h at 150 rpm at 25 ± 1 °C. In the third method, named as nAg-M3, Ag nanoparticles were loaded onto PES membrane in L-ascorbic acid solution (0.1 mol/L) at pH 2 for 24 h at 150 rpm at 25 ± 1 °C. For the preparation of nZnO coated membrane, nZnO nanoparticles solution was passed through the membrane for 24 h at 25 ± 1 °C. Anti-fouling performance of pristine and coated membranes was examined using the submerged MBR. The results showed that nZnO and nAg-M3 membranes showed lower flux decline compared with pristine membrane. Moreover, pristine and coated PES membranes were characterized using a permeation test, contact angle goniometer, and scanning electron microscopy.

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

    PubMed

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

    2014-12-11

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

  14. Electrospun superhydrophobic membranes with unique structures for membrane distillation.

    PubMed

    Liao, Yuan; Loh, Chun-Heng; Wang, Rong; Fane, Anthony G

    2014-09-24

    With modest temperature demand, low operating pressure, and high solute rejection, membrane distillation (MD) is an attractive option for desalination, waste treatment, and food and pharmaceutical processing. However, large-scale practical applications of MD are still hindered by the absence of effective membranes with high hydrophobicity, high porosity, and adequate mechanical strength, which are important properties for MD permeation fluxes, stable long-term performance, and effective packing in modules without damage. This study describes novel design strategies for highly robust superhydrophobic dual-layer membranes for MD via electrospinning. One of the newly developed membranes comprises a durable and ultrathin 3-dimensional (3D) superhydrophobic skin and porous nanofibrous support whereas another was fabricated by electrospinning 3D superhydrophobic layers on a nonwoven support. These membranes exhibit superhydrophobicity toward distilled water, salty water, oil-in-water emulsion, and beverages, which enables them to be used not only for desalination but also for other processes. The superhydrophobic dual-layer membrane #3S-N with nanofibrous support has a competitive permeation flux of 24.6 ± 1.2 kg m(-2) h(-1) in MD (feed and permeate temperate were set as 333 and 293 K, respectively) due to the higher porosity of the nanofibrous scaffold. Meanwhile, the membranes with the nonwoven support exhibit greater mechanical strength due to this support combined with better long-term performance because of the thicker 3D superhydrophobic layers. The morphology, pore size, porosity, mechanical properties, and liquid enter pressure of water of these superhydrophobic composite membranes with two different structures are reported and compared with commercial polyvinylidene fluoride membranes.

  15. N-Acyl Homoserine Lactone-Mediated Quorum Sensing with Special Reference to Use of Quorum Quenching Bacteria in Membrane Biofouling Control

    PubMed Central

    Paul, Diby

    2014-01-01

    Membrane biofouling remains a severe problem to be addressed in wastewater treatment systems affecting reactor performance and economy. The finding that many wastewater bacteria rely on N-acyl homoserine lactone-mediated quorum sensing to synchronize their activities essential for biofilm formations; the quenching bacterial quorum sensing suggests a promising approach for control of membrane biofouling. A variety of quorum quenching compounds of both synthetic and natural origin have been identified and found effective in inhibition of membrane biofouling with much less environmental impact than traditional antimicrobials. Work over the past few years has demonstrated that enzymatic quorum quenching mechanisms are widely conserved in several prokaryotic organisms and can be utilized as a potent tool for inhibition of membrane biofouling. Such naturally occurring bacterial quorum quenching mechanisms also play important roles in microbe-microbe interactions and have been used to develop sustainable nonantibiotic antifouling strategies. Advances in membrane fabrication and bacteria entrapment techniques have allowed the implication of such quorum quenching bacteria for better design of membrane bioreactor with improved antibiofouling efficacies. In view of this, the present paper is designed to review and discuss the recent developments in control of membrane biofouling with special emphasis on quorum quenching bacteria that are applied in membrane bioreactors. PMID:25147787

  16. Smart zwitterionic membranes with on/off behavior for protein transport.

    PubMed

    Su, Yanlei; Zheng, Lili; Li, Chao; Jiang, Zhongyi

    2008-09-25

    Poly(acrylonitrile) (PAN)-based zwitterionic membranes, composed of PAN and poly( N, N-dimethyl- N-methacryloxyethyl- N-(3-sulfopropyl) copolymer, are electrolyte-sensitive smart membranes. The hydrophilicity was increased and protein adsorption was remarkably decreased for the membranes in response to environmental stimuli. FTIR spectroscopic analysis directly provided molecular-level observation of the enhanced dissociation and hydration of zwitterionic sulfobetaine dipoles at higher electrolyte concentrations. The smart PAN-based zwitterionic membranes can close or open channels for protein transport under different NaCl concentrations. The electrolyte-sensitive switch of on/off behavior for protein transport is reversible.

  17. Reorganization of plasma membrane lipid domains during conidial germination.

    PubMed

    Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

    2017-02-01

    Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Preparation and Properties of Nano-Hydroxyapatite/Gelatin/Poly(vinyl alcohol) Composite Membrane.

    PubMed

    Liao, Haotian; Shi, Kun; Peng, Jinrong; Qu, Ying; Liao, Jinfeng; Qian, Zhiyong

    2015-06-01

    In this study, the bone-like composite membrane based on blends of gelatin (Gel), nano-hydroxyapatite (n-HA) and poly(vinyl alcohol) (PVA) was fabricated by solvent casting and evaporation methods. The effect of n-HA content and the ratio of Gel/PVA on the properties of the composite was investigated. The Gel/PVA and n-HA/Gel/PVA composite membranes were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), water contact angle measurement and scanning electron microscopy (SEM). The mechanical properties of the composites were determined by tensile tests. The as prepared composite membranes exhibited hydrophobility, the water contact angle of composite membrane was 126.6 when its mass ratio of n-HA/Gel/PVA was 10/50/40. The tensile strength of composite membranes was greatly increased due to the introduction of n-HA, and the tensile strength was increased to 74.92 MPa when the mass ratio of n-HA/Gel/PVA was 10/50/40. SEM observation indicated that n-HA was dispersed in the membranes and a sea-island structure was formed in the n-HA/Gel/PVA composite membranes, resulting in a significant increase in tensile strength. The as-prepared n-HA/Gel/PVA composite membranes may be applied in the field of bone tissue engineering.

  19. Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein.

    PubMed

    Li, J; Quilty, J; Popov, M; Reithmeier, R A

    2000-07-01

    The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn(642) in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.

  20. Identification and biochemical characterization of an acid sphingomyelinase-like protein from the bacterial plant pathogen Ralstonia solanacearum that hydrolyzes ATP to AMP but not sphingomyelin to ceramide.

    PubMed

    Airola, Michael V; Tumolo, Jessica M; Snider, Justin; Hannun, Yusuf A

    2014-01-01

    Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.

  1. Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering*

    PubMed Central

    Heinrich, Frank; Nanda, Hirsh; Goh, Haw Zan; Bachert, Collin; Lösche, Mathias; Linstedt, Adam D.

    2014-01-01

    The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. PMID:24505136

  2. Nature and mechanism of action of the CAMP protein of group B streptococci.

    PubMed Central

    Bernheimer, A W; Linder, R; Avigad, L S

    1979-01-01

    The extracellular product of group B streptococci responsible for the CAMP reaction was purified to near homogeneity. It is a relatively thermostable protein having a molecular weight of 23,500 and an isoelectric pH of 8.3. It was found that the CAMP reaction could be simulated by substituting [14C]glucose-containing liposomes prepared from sphingomyelin, cholesterol, and dicetyl phosphate for sheep erythrocytes. In the belief that the liposome system is a valid model, the mechanism of the CAMP reaction was further investigated by using liposomes in which N-acylsphingosine (ceramide) was substituted for sphingomyelin. In this system disruption of liposomes, as measured by release of trapped [14C]glucose, was effected by CAMP protein alone. As judged from thin-layer chromatography, CAMP protein caused no reduction in the amount of ceramide present in ceramide-containing liposomes, nor were split products demonstrable. However, binding of CAMP protein to ceramide-containing liposomes could be shown. It is inferred that in sheep erythrocytes CAMP protein reacts nonenzymatically with membrane ceramide formed by the prior action of staphylococcal sphingomyelinase and that binding of CAMP protein to ceramide disorganizes the lipid bilayer to an extent that results in cell lysis. Images PMID:378839

  3. Epithelial Membrane Protein-2 in Human Proliferative Vitreoretinopathy and Epiretinal Membranes.

    PubMed

    Telander, David G; Yu, Alfred K; Forward, Krisztina I; Morales, Shawn A; Morse, Lawrence S; Park, Susanna S; Gordon, Lynn K

    2016-06-01

    To determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target. Preretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic. All of the membranes stained positive for EMP2. Proliferative vitreoretinopathy-induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested. All studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.

  4. Enhanced photodynamic activity of hypericin by penetration enhancer N-methyl pyrrolidone formulations in the chick chorioallantoic membrane model.

    PubMed

    Saw, Constance Lay Lay; Heng, Paul Wan Sia; Chin, William Wei Lim; Soo, Khee Chee; Olivo, Malini

    2006-07-08

    Hypericin (HY) was examined for photodynamic therapy (PDT)-induced vascular damage using the chick chorioallantoic membrane (CAM) model. Clinically, plasma protein was used to solubilize HY. Upon binding to albumin, free HY available to be transported through the membrane may be limited. Hence, formulations containing a biocompatible solvent, N-Methyl pyrrolidone (NMP), have the potential to enhance HY delivery into solid tumors. At suitable concentrations, NMP and/or light irradiation did not produce antivascular damage. Hypericin-PDT effects showed to be HY and NMP concentrations-dependent. These findings indicate that NMP is a promising solvent and penetration enhancer for HY-PDT clinical applications.

  5. Nanocomposite membranes based on polybenzimidazole and ZrO2 for high-temperature proton exchange membrane fuel cells.

    PubMed

    Nawn, Graeme; Pace, Giuseppe; Lavina, Sandra; Vezzù, Keti; Negro, Enrico; Bertasi, Federico; Polizzi, Stefano; Di Noto, Vito

    2015-04-24

    Owing to the numerous benefits obtained when operating proton exchange membrane fuel cells at elevated temperature (>100 °C), the development of thermally stable proton exchange membranes that demonstrate conductivity under anhydrous conditions remains a significant goal for fuel cell technology. This paper presents composite membranes consisting of poly[2,2'-(m-phenylene)-5,5'-bibenzimidazole] (PBI4N) impregnated with a ZrO2 nanofiller of varying content (ranging from 0 to 22 wt %). The structure-property relationships of the acid-doped and undoped composite membranes have been studied using thermogravimetric analysis, differential scanning calorimetry, dynamic mechanical analysis, wide-angle X-ray scattering, infrared spectroscopy, and broadband electrical spectroscopy. Results indicate that the level of nanofiller has a significant effect on the membrane properties. From 0 to 8 wt %, the acid uptake as well as the thermal and mechanical properties of the membrane increase. As the nanofiller level is increased from 8 to 22 wt % the opposite effect is observed. At 185 °C, the ionic conductivity of [PBI4N(ZrO2 )0.231 ](H3 PO4 )13 is found to be 1.04×10(-1)  S cm(-1) . This renders membranes of this type promising candidates for use in high-temperature proton exchange membrane fuel cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Two-Ply Composite Membranes with Separation Layers from Chitosan and Sulfoethylcellulose on a Microporous Support Based on Poly(diphenylsulfone-N-phenylphthalimide).

    PubMed

    Kononova, Svetlana V; Kruchinina, Elena V; Petrova, Valentina A; Baklagina, Yulia G; Romashkova, Kira A; Orekhov, Anton S; Klechkovskaya, Vera V; Skorik, Yury A

    2017-12-14

    Two-ply composite membranes with separation layers from chitosan and sulfoethylcellulose were developed on a microporous support based on poly(diphenylsulfone- N -phenylphthalimide) and investigated by use of X-ray diffraction and scanning electron microscopy methods. The pervaporation properties of the membranes were studied for the separation of aqueous alcohol (ethanol, propan-2-ol) mixtures of different compositions. When the mixtures to be separated consist of less than 15 wt % water in propan-2-ol, the membranes composed of polyelectrolytes with the same molar fraction of ionogenic groups (-NH₃⁺ for chitosan and -SO₃ - for sulfoethylcellulose) show high permselectivity (the water content in the permeate was 100%). Factors affecting the structure of a non-porous layer of the polyelectrolyte complex formed on the substrate surface and the contribution of that complex to changes in the transport properties of membranes are discussed. The results indicate significant prospects for the use of chitosan and sulfoethylcellulose for the formation of highly selective pervaporation membranes.

  7. Internal Membrane Control in Azotobacter vinelandii

    PubMed Central

    Pate, Jack L.; Shah, Vinod K.; Brill, Winston J.

    1973-01-01

    Azotobacter vinelandii was grown on N2, NH4+, or NO3−, and an internal membrane network was observed by electron microscopy of thin sections of cells. Cells obtained in early exponential growth contained less internal membrane than did cells from cultures in late exponential growth. It seems likely that O2 has a role in regulating the amount of internal membrane structure. Images PMID:4123239

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

    NASA Astrophysics Data System (ADS)

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

    2018-05-01

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

  9. NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

    PubMed

    Eisenbeis, Simone; Lohmiller, Stefanie; Valdebenito, Marianne; Leicht, Stefan; Braun, Volkmar

    2008-08-01

    Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

  10. Membrane Permeabilization Induced by Sphingosine: Effect of Negatively Charged Lipids

    PubMed Central

    Jiménez-Rojo, Noemi; Sot, Jesús; Viguera, Ana R.; Collado, M. Isabel; Torrecillas, Alejandro; Gómez-Fernández, J.C.; Goñi, Félix M.; Alonso, Alicia

    2014-01-01

    Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease. PMID:24940775

  11. Desolventizing of soybean oil/azeotrope mixtures using ceramic membranes.

    PubMed

    de Melo, Jonas R M; Tiggeman, Lidia; Rezzadori, Katia; Steffens, Juliana; Palliga, Marshall; Oliveira, J Vladimir; Di Luccio, Marco; Tres, Marcus V

    2017-08-01

    This work investigates the use of ceramic membranes with different molecular weight cut-offs (MWCOs: 5, 10 and 20 kDa) to desolventize azeotropic solvent mixtures (ethanol/n-hexane and isopropyl alcohol/n-hexane) from soybean oil/azeotrope micelles. Results show that a decrease in the MWCO of a membrane and an increase in the solvent mass ratio in the mixture resulted in a significant reduction in the permeate flux. The 20 kDa membrane presented the highest permeate flux, 80 and 60 kg/m 2 h for the soybean oil/n-hexane/isopropyl alcohol and soybean oil/n-hexane/ethanol azeotropes, respectively, for an oil to solvent ratio of 1:3 (w/w). The highest oil retention was found using the n-hexane/isopropyl alcohol azeotrope, around 25% in the membrane with the lowest MWCO, that is, 5 kDa. It is shown that the azeotropic mixtures provided intermediate characteristics compared to the original pure solvent behavior.

  12. Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes.

    PubMed

    Sakuma, Shinji; Suita, Masaya; Yamamoto, Takafumi; Masaoka, Yoshie; Kataoka, Makoto; Yamashita, Shinji; Nakajima, Noriko; Shinkai, Norihiro; Yamauchi, Hitoshi; Hiwatari, Ken-Ichiro; Hashizume, Akio; Tachikawa, Hiroyuki; Kimura, Ryoji; Ishimaru, Yuki; Kasai, Atsushi; Maeda, Sadaaki

    2012-05-01

    We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30 min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120 min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Molecular mechanism for sphingosine-induced Pseudomonas ceramidase expression through the transcriptional regulator SphR

    PubMed Central

    Okino, Nozomu; Ito, Makoto

    2016-01-01

    Pseudomonas aeruginosa, an opportunistic, but serious multidrug-resistant pathogen, secretes a ceramidase capable of cleaving the N-acyl linkage of ceramide to generate fatty acids and sphingosine. We previously reported that the secretion of P. aeruginosa ceramidase was induced by host-derived sphingolipids, through which phospholipase C-induced hemolysis was significantly enhanced. We herein investigated the gene(s) regulating sphingolipid-induced ceramidase expression and identified SphR, which encodes a putative AraC family transcriptional regulator. Disruption of the sphR gene in P. aeruginosa markedly decreased the sphingomyelin-induced secretion of ceramidase, reduced hemolytic activity, and resulted in the loss of sphingomyelin-induced ceramidase expression. A microarray analysis confirmed that sphingomyelin significantly induced ceramidase expression in P. aeruginosa. Furthermore, an electrophoretic mobility shift assay revealed that SphR specifically bound free sphingoid bases such as sphingosine, dihydrosphingosine, and phytosphingosine, but not sphingomyelin or ceramide. A β-galactosidase-assisted promoter assay showed that sphingosine activated ceramidase expression through SphR at a concentration of 100 nM. Collectively, these results demonstrated that sphingosine induces the secretion of ceramidase by promoting the mRNA expression of ceramidase through SphR, thereby enhancing hemolytic phospholipase C-induced cytotoxicity. These results facilitate understanding of the physiological role of bacterial ceramidase in host cells. PMID:27941831

  14. First Implantation of Silicon Nanopore Membrane Hemofilters

    PubMed Central

    Kensinger, Clark; Karp, Seth; Kant, Rishi; Chui, Benjamin W.; Goldman, Kenneth; Yeager, Torin; Gould, Edward R.; Buck, Amanda; Laneve, David C.; Groszek, Joseph J.; Roy, Shuvo; Fissell, William H.

    2016-01-01

    An implantablehemofilter for the treatment of kidney failure depends critically on the transport characteristics of the membrane and the biocompatibility of the membrane, cartridge, and blood conduits. A novel membrane with slit-shaped pores optimizes the trade-off between permeability and selectivity, enabling implanted therapy. Sustained (3–8) day function of an implanted parallel-plate hemofilter with minimal anticoagulation was achieved by considering biocompatibility at the subnanometer scale of chemical interactions and the millimeter scale of blood fluid dynamics. A total of 400 nm-thick polysilicon flat sheet membranes with 5–8 nm 2 micron slit-shaped pores were surface-modified with polyethylene glycol. Hemofilter cartridge geometries were refined based on computational fluid dynamics predictions of blood flow. In an uncontrolled pilot study, silicon filters were implanted in six class A dogs. Cartridges were connected to the cardiovascular system by anastamoses to the aorta and inferior vena cava and filtrate was drained to collection pouches positioned in the peritoneum. Pain medicine and acetylsalicylic acid were administered twice daily until the hemofilters were harvested on postoperative days 3 (n = 2), 4 (n = 2), 5 (n = 1), and 8 (n = 1). No hemofilters were thrombosed. Animals treated for 5 and 8 days had microscopic fractures in the silicon nanopore membranes and 20–50 ml of transudative (albumin sieving coefficient 0.5 – 0.7) fluid in the collection pouches at the time of explant. Shorter experimental durations (3–4 days) resulted in filtration volumes similar to predictions based on mean arterial pressures and membrane hydraulic permeability and (∼ 0.2 – 0.3), similar to preimplantation measurements. In conclusion, a detailed mechanistic and materials science attention to blood–material interactions allows implanted hemofilters to resist thrombosis. Additional testing is needed to determine optimal membrane characteristics and

  15. Biosynthetic elongation of isolated teichuronic acid polymers via glucosyl- and N-acetylmannosaminuronosyltransferases from solubilized cytoplasmic membrane fragments of Micrococcus luteus.

    PubMed Central

    Hildebrandt, K M; Anderson, J S

    1990-01-01

    Cytoplasmic membrane fragments of Micrococcus luteus catalyze in vitro biosynthesis of teichuronic acid from uridine diphosphate D-glucose (UDP-glucose), uridine diphosphate N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), and uridine diphosphate N-acetyl-D-glucosamine. Membrane fragments solubilized with Thesit (dodecyl alcohol polyoxyethylene ether) can utilize UDP-glucose and UDP-ManNAcA to effect elongation of teichuronic acid isolated from native cell walls. When UDP-glucose is the only substrate supplied, the detergent-solubilized glucosyltransferase incorporates a single glucosyl residue onto each teichuronic acid acceptor. When both UDP-glucose and UDP-ManNAcA are supplied, the glucosyltransferase and the N-acetylmannosaminuronosyltransferase act cooperatively to elongate the teichuronic acid acceptor by multiple additions of the disaccharide repeat unit. As shown by polyacrylamide gel electrophoresis, low-molecular-weight fractions of teichuronic acid are converted to higher-molecular-weight polymers by the addition of as many as 17 disaccharide repeat units. Images PMID:2118507

  16. Analysis of Nitrification Efficiency and Microbial Community in a Membrane Bioreactor Fed with Low COD/N-Ratio Wastewater

    PubMed Central

    Ma, Jinxing; Wang, Zhiwei; Zhu, Chaowei; Liu, Shumeng; Wang, Qiaoying; Wu, Zhichao

    2013-01-01

    In this study, an approach using influent COD/N ratio reduction was employed to improve process performance and nitrification efficiency in a membrane bioreactor (MBR). Besides sludge reduction, membrane fouling alleviation was observed during 330 d operation, which was attributed to the decreased production of soluble microbial products (SMP) and efficient carbon metabolism in the autotrophic nitrifying community. 454 high-throughput 16S rRNA gene pyrosequencing revealed that the diversity of microbial sequences was mainly determined by the feed characteristics, and that microbes could derive energy by switching to a more autotrophic metabolism to resist the environmental stress. The enrichment of nitrifiers in an MBR with a low COD/N-ratio demonstrated that this condition stimulated nitrification, and that the community distribution of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) resulted in faster nitrite uptake rates. Further, ammonia oxidation was the rate-limiting step during the full nitrification. PMID:23667573

  17. The N-terminal amphipathic helix of Pex11p self-interacts to induce membrane remodelling during peroxisome fission.

    PubMed

    Su, Juanjuan; Thomas, Ann S; Grabietz, Tanja; Landgraf, Christiane; Volkmer, Rudolf; Marrink, Siewert J; Williams, Chris; Melo, Manuel N

    2018-06-01

    Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Mechanistic insights into porous graphene membranes for helium separation and hydrogen purification

    NASA Astrophysics Data System (ADS)

    Wei, Shuxian; Zhou, Sainan; Wu, Zhonghua; Wang, Maohuai; Wang, Zhaojie; Guo, Wenyue; Lu, Xiaoqing

    2018-05-01

    Porous graphene (PG) and nitrogen-substituted PG monolayers of 3N-PG and 6N-PG were designed as effective membranes for the separation of He and H2 over Ne, Ar, N2, CO, and CH4 by using density functional theory. Results showed that PG and 3N-PG exhibited suitable pore sizes and relatively high stabilities for He and H2 separation. PG and 3N-PG membranes also presented excellent He and H2 selectivities over Ne, Ar, N2, CO and CH4 at a wide temperature range. 6N-PG membrane exerted unexceptionable permeances of the studied gases, especially He and H2, which could remarkably improve the separation efficiency of He and H2. Analyses on the most stable adsorption configurations and maximum adsorption energies indicated weak Van der Waals interactions between the gases and the three PG-based membranes. Microscopic permeation process analyses based on the minimum energy pathway, energy profiles, and electron density isosurfaces elucidated the remarkable selectivities of He over Ne/CO/N2/Ar/CH4 and H2 over CO/N2/CH4 and the high permeances of He and H2 passing through the three PG-based membranes. This work not only highlighted the potential use of the three PG-based membranes for He separation and H2 purification but also provided a superior alternative strategy to design and screen membrane materials for gas separation.

  19. Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation.

    PubMed

    Chakravarthy, B R; Wong, J; Durkin, J P

    1995-10-01

    Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.

  20. Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

    PubMed Central

    Mayor, S; Sabharanjak, S; Maxfield, F R

    1998-01-01

    Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins. PMID:9707422

  1. Protein valves prepared by click reaction grafting of poly(N-isopropylacrylamide) to electrospun poly(vinyl chloride) fibrous membranes

    NASA Astrophysics Data System (ADS)

    Guo, Jian-Wei; Lin, Zhen-Yu; Chang, Chi-Jung; Lu, Chien-Hsing; Chen, Jem-Kun

    2018-05-01

    In this study, poly(vinyl chloride) (PVC) was electrospun into fibrous membranes and then reacted with NaN3 to generate azido-terminated PVC fibrous membranes. A propargyl-terminated poly(N-isopropylacrylamide) (PNIPAAm) was also synthesized and then grafted, through click reactions, onto the azido-terminated PVC fiber surface. Protrusion-, scale-, and joint-like structures of the PNIPAAm grafts on the PVC fibers were formed upon increasing the molecular weight of the PNIPAAm grafts. The PNIPAAm-grafted PVC fibrous mats exhibited completely wetted surfaces at 25 °C because of their high roughness. The static water contact angle of the PNIPAAm-grafted PVC fibrous mats reached 140° when the temperature was increased to 45 °C. This thermoresponsive behavior was significantly greater than that of the PNIPAAm grafted on a flat surface. Temperature-responsive membranes were constructed having a pore size of 1.38 μm and applied as protein valves to block and release an antibody (fluorescein-conjugated AffiniPure goat anti-rabbit IgG). At 25 °C, the collection efficiency remained at 94% for antibody concentrations up to 60 ng/L. As the temperature increased to 45 °C, the collection efficiency decreased abruptly, to 4%, when the antibody concentration was greater than 20 ng/L. Accordingly, this system of PNIPAAm-grafted PVC fibers functioned as a protein valve allowing the capture and concentration of proteins.

  2. New look inside human breast ducts with Raman imaging. Raman candidates as diagnostic markers for breast cancer prognosis: Mammaglobin, palmitic acid and sphingomyelin.

    PubMed

    Abramczyk, Halina; Brozek-Pluska, Beata

    2016-02-25

    Looking inside the human body fascinated mankind for thousands of years. Current diagnostic and therapy methods are often limited by inadequate sensitivity, specificity and spatial resolution. Raman imaging may bring revolution in monitoring of disease and treatment. The main advantage of Raman imaging is that it gives spatial information about various chemical constituents in defined cellular organelles in contrast to conventional methods (liquid chromatography/mass spectrometry, NMR, HPLC) that rely on bulk or fractionated analyses of extracted components. We demonstrated how Raman imaging can drive the progress on breast cancer just unimaginable a few years ago. We looked inside human breast ducts answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the stroma around the duct during cancer development. We have identified Raman candidates as diagnostic markers for breast cancer prognosis: carotenoids, mammaglobin, palmitic acid and sphingomyelin as key molecular targets in ductal breast cancer in situ, and propose the molecular mechanisms linking oncogenes with lipid programming. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Silicon Carbide membranes as substrate for Synchrotron measurements

    NASA Astrophysics Data System (ADS)

    Altissimo, M.; Iacopi, A.; Hold, L.; Matruglio, A.; Zucchiatti, P.; Vaccari, L.; Bedolla, D. E.; Ulloa Severino, L.; Parisse, P.; Gianoncelli, A.

    2018-05-01

    Silicon Nitride (SiN) membranes have long been the substrate of choice for many different synchrotron techniques at very different wavelengths (from IR to hard X-rays), due to their ease of production, relative robustness even in films <200 nm in thickness, and compatibility with standard microfabrication techniques. Here we present a set of data referring to custom-made Silicon Carbide (SiC) windows. We measured SiC surface roughness, mechanical robustness and membrane transmission both at IR and soft X-rays wavelengths, and compared the data with standard Si3N4, acquired in the same conditions. Further, we grew HEK293T cells both on Si3N4 and SiC membranes, and analysed them with IR and soft X-ray microscopy. Our data demonstrates how SiC is an excellent choice as membrane material for synchrotron measurements, since it shows higher transmission and higher robustness as compared to Si3N4 of the same thickness, and an improved compatibility for cell culturing, allowing to postulate their use also for bio-oriented research.

  4. Layered plasma polymer composite membranes

    DOEpatents

    Babcock, Walter C.

    1994-01-01

    Layered plasma polymer composite fluid separation membranes are disclosed, which comprise alternating selective and permeable layers for a total of at least 2n layers, where n is .gtoreq.2 and is the number of selective layers.

  5. All n-3 PUFA are not the same: MD simulations reveal differences in membrane organization for EPA, DHA and DPA.

    PubMed

    Leng, Xiaoling; Kinnun, Jacob J; Cavazos, Andres T; Canner, Samuel W; Shaikh, Saame Raza; Feller, Scott E; Wassall, Stephen R

    2018-05-01

    Eicosapentaenoic (EPA, 20:5), docosahexaenoic (DHA, 22:6) and docosapentaenoic (DPA, 22:5) acids are omega-3 polyunsaturated fatty acids (n-3 PUFA) obtained from dietary consumption of fish oils that potentially alleviate the symptoms of a range of chronic diseases. We focus here on the plasma membrane as a site of action and investigate how they affect molecular organization when taken up into a phospholipid. All atom MD simulations were performed to compare 1-stearoyl-2-eicosapentaenoylphosphatylcholine (EPA-PC, 18:0-20:5PC), 1-stearoyl-2-docosahexaenoylphosphatylcholine (DHA-PC, 18:0-22:6PC), 1-stearoyl-2-docosapentaenoylphosphatylcholine (DPA-PC, 18:0-22:5PC) and, as a monounsaturated control, 1-stearoyl-2-oleoylphosphatidylcholine (OA-PC, 18:0-18:1PC) bilayers. They were run in the absence and presence of 20mol% cholesterol. Multiple double bonds confer high disorder on all three n-3 PUFA. The different number of double bonds and chain length for each n-3 PUFA moderates the reduction in membrane order exerted (compared to OA-PC, S¯ CD =0.152). EPA-PC (S¯ CD =0.131) is most disordered, while DPA-PC (S¯ CD =0.140) is least disordered. DHA-PC (S¯ CD =0.139) is, within uncertainty, the same as DPA-PC. Following the addition of cholesterol, order in EPA-PC (S¯ CD =0.169), DHA-PC (S¯ CD =0.178) and DPA-PC (S¯ CD =0.182) is increased less than in OA-PC (S¯ CD =0.214). The high disorder of n-3 PUFA is responsible, preventing the n-3 PUFA-containing phospholipids from packing as close to the rigid sterol as the monounsaturated control. Our findings establish that EPA, DHA and DPA are not equivalent in their interactions within membranes, which possibly contributes to differences in clinical efficacy. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented 15N and 31P Solid-State NMR Spectroscopy

    PubMed Central

    Verly, Rodrigo M.; Moraes, Cléria Mendonça de; Resende, Jarbas M.; Aisenbrey, Christopher; Bemquerer, Marcelo Porto; Piló-Veloso, Dorila; Valente, Ana Paula; Almeida, Fábio C.L.; Bechinger, Burkhard

    2009-01-01

    DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing. PMID:19289046

  7. Layered plasma polymer composite membranes

    DOEpatents

    Babcock, W.C.

    1994-10-11

    Layered plasma polymer composite fluid separation membranes are disclosed, which comprise alternating selective and permeable layers for a total of at least 2n layers, where n is [>=]2 and is the number of selective layers. 2 figs.

  8. Performance and membrane fouling of a step-fed submerged membrane sequencing batch reactor treating swine biogas digestion slurry.

    PubMed

    Han, Zhiying; Chen, Shixia; Lin, Xiaochang; Yu, Hongjun; Duan, Li'an; Ye, Zhangying; Jia, Yanbo; Zhu, Songming; Liu, Dezhao

    2018-01-02

    To identify the performance of step-fed submerged membrane sequencing batch reactor (SMSBR) treating swine biogas digestion slurry and to explore the correlation between microbial metabolites and membrane fouling within this novel reactor, a lab-scale step-fed SMSBR was operated under nitrogen loading rate of 0.026, 0.052 and 0.062 g NH 4 + -N (gVSS·d) -1 . Results show that the total removal efficiencies for NH 4 + -N, total nitrogen and chemical oxygen demand in the reactor (>94%, >89% and >97%, respectively) were high during the whole experiment. However, the cycle removal efficiency of NH 4 + -N decreased significantly when the nitrogen loading rate was increased to 0.062 g NH 4 + -N (gVSS·d) -1 . The total removal efficiency of total phosphorus in the step-fed SMSBR was generally higher than 75%, though large fluctuations were observed during the experiments. In addition, the concentrations of microbial metabolites, i.e., soluble microbial products (SMP) and extracellular polymeric substances (EPS) from activated sludge increased as nitrogen loading rate increased, both showing quadratic equation correlations with viscosity of the mixed liquid in the step-fed SMSBR (both R 2 > 0.90). EPS content was higher than SMP content, while protein (PN) was detected as the main component in both SMP and EPS. EPS PN was found to be well correlated with transmembrane pressure, membrane flux and the total membrane fouling resistance. Furthermore, the three-dimensional excitation-emission matrix fluorescence spectroscopy results suggested the tryptophan-like protein as one of the main contributors to the membrane fouling. Overall, this study showed that the step-fed SMSBR could be used to treat swine digestion slurry at nitrogen loading rate of 0.052 g NH 4 + -N (gVSS·d) -1 , and the control strategy of membrane fouling should be developed based on reducing the tryptophan-like PN in EPS.

  9. Membrane Restructuring Events during the Enzymatic Generation of Ceramides with Very Long-Chain Polyunsaturated Fatty Acids.

    PubMed

    Peñalva, Daniel A; Antollini, Silvia S; Ambroggio, Ernesto E; Aveldaño, Marta I; Fanani, María L

    2018-04-10

    In rat sperm heads, sphingomyelin (SM) species that contain very long-chain polyunsaturated fatty acid (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalyzed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs)) containing phosphatidylcholine, and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated and more rapidly than P-Cer. Nephelometry and dynamic light scattering showed that LUVs tended to form large lipid particles more intensely, and Förster resonance energy transfer (FRET) increases suggested that lateral lipid mixing was more marked when V-Cer rather than P-Cer was produced. As reported by 6-dodecanoyl-2-dimethyl-aminopnaphthalene (Laurdan) and 1,6-diphenyl-1,3,5,-hexatriene (DPH), the production of V-Cer resulted in higher and faster restriction in lipid mobility than that of P-Cer, implying a stronger increase in membrane dehydration and microviscosity. Moreover, DPH anisotropy suggested a higher solubility of V-Cer than that of P-Cer in the liquid-disordered phase. At room temperature, liquid-condensed lateral domains appeared in P-SM- but not in V-SM-containing GUVs. The former maintained their size while losing their contents gradually during SMase action, whereas the latter became permeable earlier and reduced their size in few minutes until suddenly collapsing. The fast and potent generation of V-Cer may contribute to the membrane restructuring events that occur on the acrosome-reacted sperm head.

  10. Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

    PubMed

    Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

    2014-04-22

    Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

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

    PubMed Central

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

    2013-01-01

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

  12. Hydrostatic and Flow Measurements on Wrinkled Membrane Walls

    NASA Astrophysics Data System (ADS)

    Ozsun, Ozgur; Ekinci, Kamil

    2013-03-01

    In this study, we investigate structural properties of wrinkled silicon nitride (SiN) membranes, under both hydrostatic perturbations and flow conditions, through surface profile measurements. Rectangular SiN membranes with linear dimensions of 15 mm × 1 . 5 mm × 1 μ m are fabricated on a 500 - μ m-thick silicon substrate using standard lithography techniques. These thin, initially flat, tension-dominated membranes are wrinkled by bending the silicon substrate. The wrinkled membranes are subsequently incorporated as walls into rectangular micro-channels, which allow both hydrostatic and flow measurements. The structural response of the wrinkles to hydrostatic pressure provides a measure of the various energy scales in the problem. Flow experiments show that the elastic properties and the structural undulations on a compliant membrane completely dominate the flow, possibly providing drag reduction. These measurements pave the way for building and using compliant walls for drag reduction in micro-channels.

  13. Single crystal diamond membranes for nanoelectronics.

    PubMed

    Bray, Kerem; Kato, Hiromitsu; Previdi, Rodolfo; Sandstrom, Russell; Ganesan, Kumaravelu; Ogura, Masahiko; Makino, Toshiharu; Yamasaki, Satoshi; Magyar, Andrew P; Toth, Milos; Aharonovich, Igor

    2018-02-22

    Single crystal, nanoscale diamond membranes are highly sought after for a variety of applications including nanophotonics, nanoelectronics and quantum information science. However, so far, the availability of conductive diamond membranes has remained an unreachable goal. In this work we present a complete nanofabrication methodology for engineering high aspect ratio, electrically active single crystal diamond membranes. The membranes have large lateral directions, exceeding ∼500 × 500 μm 2 and are only several hundreds of nanometers thick. We further realize vertical single crystal p-n junctions made from the diamond membranes that exhibit onset voltages of ∼10 V and a current of several mA. Moreover, we deterministically introduce optically active color centers into the membranes, and demonstrate for the first time a single crystal nanoscale diamond LED. The robust and scalable approach to engineer the electrically active single crystal diamond membranes offers new pathways for advanced nanophotonic, nanoelectronic and optomechanical devices employing diamond.

  14. Erythrocyte membrane n-3 fatty acid levels and carotid atherosclerosis in Chinese men and women.

    PubMed

    Dai, Xiao-wei; Zhang, Bo; Wang, Ping; Chen, Chao-gang; Chen, Yu-ming; Su, Yi-xiang

    2014-01-01

    Prospective studies have supported the beneficial effects of n-3 fatty acid consumption on cardiac deaths, but limited data focused on atherosclerosis. We investigated the associations between n-3 fatty acids in erythrocytes and atherosclerosis in middle-aged and older Chinese. 847 subjects (285 men and 562 women), aged 40-65 years, from Guangzhou, China were included in this community-based cross-sectional study between December 2005 and January 2008. The levels of α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in erythrocytes were measured by gas chromatography. Carotid ultrasound examination was conducted to obtain intima-media thickness of the common carotid artery and the carotid bifurcation. Dietary data and other covariates were collected using interviewer-administered questionnaires. After adjustment for age, sex, and other confounders, negative dose-response associations between the contents of individual n-3 polyunsaturated fatty acids in the erythrocyte membrane and the prevalence of carotid artery wall thickening and plaque were observed. A comparison in the highest and lowest tertiles gave odds ratios (95% confidence interval) for thickening in the walls of the common carotid artery of 0.58 (0. 34-0.97; P-trend = 0. 037) for DHA, and 0.39 (0.23-0.67; P-trend < 0.001) for ALA. However, EPA was not significantly associated with carotid atherosclerosis. Similar results were found for thickening at the carotid bifurcation and the occurrence of carotid artery plaque. Higher levels of DHA and ALA in the erythrocyte membrane were significantly associated with a lower burden of subclinical atherosclerosis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Silicon carbide ceramic membranes

    NASA Astrophysics Data System (ADS)

    Suwanmethanond, Varaporn

    This dissertation focuses on the preparation of silicon carbide (SiC) ceramic membranes on SiC substrates. An original technique of SiC porous substrate preparation using sintering methods was developed during the work for the completion of the dissertation. The resulting SiC substrates have demonstrated high porosity, high internal surface area, well interconnected surface pore network and, at the same time, good thermal, chemical and mechanical stability. In a further development, sol-gel techniques were used to deposit micro-porous SiC membranes on these SiC porous substrates. The SiC membranes were characterized by a variety of techniques: ideal gas selectivity (He and N2), XRD, BET, SEM, XPS, and AFM. The characterization results confirmed that the asymmetric sol-gel SiC membranes were of high quality, with no cracks or pinholes, and exhibiting high resistance to corrosion and high hydro-thermal stability. In conclusion, the SiC ceramic membrane work was successfully completed. Two publications in international peer reviewed journals resulted out of this work.

  16. Efficient hydrogen isotopologues separation through a tunable potential barrier: The case of a C2N membrane.

    PubMed

    Qu, Yuanyuan; Li, Feng; Zhao, Mingwen

    2017-05-03

    Isotopes separation through quantum sieving effect of membranes is quite promising for industrial applications. For the light hydrogen isotopologues (eg. H 2 , D 2 ), the confinement of potential wells in porous membranes to isotopologues was commonly regarded to be crucial for highly efficient separation ability. Here, we demonstrate from first-principles that a potential barrier is also favorable for efficient hydrogen isotopologues separation. Taking an already-synthesized two-dimensional carbon nitride (C 2 N-h2D) as an example, we predict that the competition between quantum tunneling and zero-point-energy (ZPE) effects regulated by the tensile strain leads to high selectivity and permeance. Both kinetic quantum sieving and equilibrium quantum sieving effects are considered. The quantum effects revealed in this work offer a prospective strategy for highly efficient hydrogen isotopologues separation.

  17. Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane

    PubMed Central

    Rucks, Elizabeth A.; Olson, Macy G.; Jorgenson, Lisa M.; Srinivasan, Rekha R.; Ouellette, Scot P.

    2017-01-01

    Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H2O2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncATM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncATM-APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known

  18. Molecular dynamics insights into the structural and diffusive properties of ZIF-8/PDMS mixed matrix membranes in the n-butanol/water pervaporation process

    NASA Astrophysics Data System (ADS)

    Sun, Tao; Fang, Manquan; Wu, Zhen; Yu, Lixin; Li, Jiding

    2017-04-01

    Molecular dynamics (MD) simulation was used to study the structural and diffusive properties of zeolitic imidazolate framework-8 (ZIF-8)/polydimethylsiloxane (PDMS), a novel alcohol-permselective mixed matrix membrane (MMM). Simulation models of one pure PDMS membrane and three ZIF-8/PDMS MMMs with increasing loadings were successfully constructed. Non-bond energy turned out to be a strong attractive interaction between the PDMS matrix and ZIF-8 cells. The morphology and mobility of PDMS chains were characterized by mean square displacement (MSD). The fraction of free volume (FFV) of the pure membrane and MMMs was calculated and showed declining trends with increasing ZIF-8 loadings. The diffusion coefficients of n-butanol and water molecules were calculated by the Einstein relation. {D}n-\\text{butanol} first increased then decreased, while {D}{{water}} decreased with the increasing loadings. The mechanism of selective diffusion behaviour was investigated and it was found that the inner channels of ZIF-8 provided selective pathways for n-butanol. Diffusion coefficients were correlated with FFV and the results showed that the logarithm of {D}{{water}} demonstrated a good linear relation with the inverse FFV and was in agreement with the free volume theory, while {D}n-\\text{butanol} showed a significant deviation in the case of MMM-1 due to the selective diffusion channels provided by ZIF-8.

  19. CO2 Acquisition Membrane (CAM)

    NASA Technical Reports Server (NTRS)

    Mason, Larry W.; Way, J. Douglas; Vlasse, Marcus

    2003-01-01

    The objective of CAM is to develop, test, and analyze thin film membrane materials for separation and purification of carbon dioxide (CO2) from mixtures of gases, such as those found in the Martian atmosphere. The membranes are targeted toward In Situ Resource Utilization (ISRU) applications that will operate in extraterrestrial environments and support future unmanned and human space missions. A primary application is the Sabatier Electrolysis process that uses Mars atmosphere CO2 as raw material for producing water, oxygen, and methane for rocket fuel and habitat support. Other applications include use as an inlet filter to collect and concentrate Mars atmospheric argon and nitrogen gases for habitat pressurization, and to remove CO2 from breathing gases in Closed Environment Life Support Systems (CELSS). CAM membrane materials include crystalline faujasite (FAU) zeolite and rubbery polymers such as silicone rubber (PDMS) that have been shown in the literature and via molecular simulation to favor adsorption and permeation of CO2 over nitrogen and argon. Pure gas permeation tests using commercial PDMS membranes have shown that both CO2 permeance and the separation factor relative to other gases increase as the temperature decreases, and low (Delta)P(Sub CO2) favors higher separation factors. The ideal CO2/N2 separation factor increases from 7.5 to 17.5 as temperature decreases from 22 C to -30 C. For gas mixtures containing CO2, N2, and Ar, plasticization decreased the separation factors from 4.5 to 6 over the same temperature range. We currently synthesize and test our own Na(+) FAU zeolite membranes using standard formulations and secondary growth methods on porous alumina. Preliminary tests with a Na(+) FAU membrane at 22 C show a He/SF6 ideal separation factor of 62, exceeding the Knudsen diffusion selectivity by an order of magnitude. This shows that the membrane is relatively free from large defects and associated non-selective (viscous flow) transport

  20. Synchrotron Radiation Damage Mechanism of X-Ray Mask Membranes Irradiated in Helium Environment

    NASA Astrophysics Data System (ADS)

    Arakawa, Tomiyuki; Okuyama, Hiroshi; Okada, Koichi; Nagasawa, Hiroyuki; Syoki, Tsutomu; Yamaguchi, Yoh-ichi

    1992-12-01

    The mechanism of X-ray mask membrane displacement induced by synchrotron radiation (SR) has been discussed. Silicon nitride (SiN) and silicon carbide (SiC) membranes were irradiated by SR in a 1 atm helium ambient. SR-induced displacement for both membranes was 25-97 nm (σ). Oxygen concentration in both SiN and SiC was below 0.01 in O/Si atomic ratio. Although an increase in dangling bond density of SiN was observed, no remarkable increase in spin density was detected in SiC. Moreover, the most important finding was that thin oxides were grown on the membrane surface after SR irradiation. From these results, it is considered that the oxide growth on SiC membrane surfaces, and both the oxide growth and the increase of dangling bond density in SiN play an important role in the SR-induced displacement for the X-ray mask membranes.

  1. Bilayer registry in a multicomponent asymmetric membrane: Dependence on lipid composition and chain length

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

    Polley, Anirban; Mayor, Satyajit; Rao, Madan, E-mail: madan@rri.res.in, E-mail: madan@ncbs.res.in

    2014-08-14

    A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component asymmetric lipid bilayer comprising an unsaturated palmitoyl-oleoyl-phosphatidyl-choline, a saturated sphingomyelin, and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered (l{sub o})-liquid disorderedmore » (l{sub d}) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed l{sub d} phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the l{sub o} domains across the two leaflets, resulting in bilayer registry. This transbilayer correlation depends sensitively upon the chain length of the participating lipids and possibly other features of lipid chemistry, such as degree of saturation. We find that the l{sub o} domains in the upper leaflet can induce phase segregation in the lower leaflet, when the latter is nominally in the mixed (l{sub d}) phase.« less

  2. Polyethersulfone/polyacrylonitrile blended ultrafiltration membranes: preparation, morphology and filtration properties.

    PubMed

    Pasaoglu, Mehmet Emin; Guclu, Serkan; Koyuncu, Ismail

    Polyethersulfone (PES)/polyacrylonitrile (PAN) membranes have been paid attention among membrane research subjects. However, very few studies are included in the literature. In our study, asymmetric ultrafiltration (UF) membranes were prepared from blends of PES/PAN with phase inversion method using water as coagulation bath. Polyvinylpyrrolidone (PVP) with Mw of 10,000 Da was used as pore former agent. N,N-dimethylformamide was used as solvent. The effects of different percentage of PVP and PES/PAN composition on morphology and water filtration properties were investigated. Membrane performances were examined using pure water and lake water filtration studies. Performances of pure water were less with the addition of PAN into the PES polymer casting solutions. However, long-term water filtration tests showed that PES/PAN blend membranes anti-fouling properties were much higher than the neat PES membranes. The contact angles of PES/PAN membranes were lower than neat PES membranes because of PAN addition in PES polymer casting solutions. Furthermore, it was found that PES/PAN blend UF membranes' dynamic mechanical analysis properties in terms of Young's modules were less than neat PES membrane because of decreasing amount of PES polymer.

  3. Modulation of N-glycosylation by mesalamine facilitates membranous E-cadherin expression in colon epithelial cells☆

    PubMed Central

    Khare, Vineeta; Lang, Michaela; Dammann, Kyle; Campregher, Christoph; Lyakhovich, Alex; Gasche, Christoph

    2014-01-01

    Genome wide association studies have implicated intestinal barrier function genes in the pathogenesis of ulcerative colitis. One of such loci CDH1, encoding E-cadherin, a transmembrane glycoprotein with known tumor suppressor functions, is also linked to the susceptibility to colorectal cancer. Loss of membranous E-cadherin expression is common in both colitis and cancer. We have recently demonstrated that mesalamine (5-ASA); the anti-inflammatory drug used to treat ulcerative colitis, induces membranous expression of E-cadherin and increases intercellular adhesion. Using colorectal cancer epithelial cells with aberrant E-cadherin expression, we investigated the mechanism underlying such an effect of 5-ASA. Post-translational modification of E-cadherin glycosylation was analyzed by biotin/streptavidin detection of sialylated glycoproteins. GnT-III (N-acetylglucosaminyltransferase III) expression was assessed by qRT-PCR, Western blot and immunofluorescence. GnT-III activity was analyzed by reactivity with E-4/L-4-PHA. Expression, localization and interaction of E-cadherin and β-catenin were analyzed by Western blot, immunocytochemistry and RNA interference. 5-ASA activity modulated E-cadherin glycosylation and increased both mRNA and protein levels of GnT-III and its activity as detected by increased E4-lectin reactivity. Intestinal APCMin polyps in mice showed low expression of GnT-III and 5-ASA was effective in increasing its expression. The data demonstrated that remodeling of glycans by GnT-III mediated bisect glycosylation, contributes to the membranous retention of E-cadherin by 5-ASA; facilitating intercellular adhesion. Induction of membranous expression of E-cadherin by 5-ASA is a novel mechanism for mucosal healing in colitis that might impede tumor progression by modulation of GnT-III expression. PMID:24184502

  4. A nitrogen-doped carbon nanotube enhanced polyethersulfone membrane system for water treatment

    NASA Astrophysics Data System (ADS)

    Phao, Neo; Nxumalo, Edward N.; Mamba, Bhekie B.; Mhlanga, Sabelo D.

    Water quality in South Africa and around the world continues to deteriorate due to contamination by organic, inorganic and microbial substances. While many efforts have been done to address water quality problems, current drinking water treatment technologies remain costly and do not effectively remove pollutants to acceptable levels. In this work nitrogen doped carbon nanotubes/polyethersulfone (N-CNT/PES) blend membranes were synthesized via a modified phase inversion method and assessed for suitability in drinking water treatment. The N-CNTs with outer diameters of 30-45 nm and 3% N content were prepared using a conventional chemical vapour deposition method and functionalized by refluxing in HNO3. The confirmation and degree of functionalization with -OH and -COOH groups was determined using Fourier-transform infrared (FTIR) spectroscopy and zeta potential analysis. FTIR studies confirmed the successful incorporation of functionalized N-CNTs (N-CNTs) in the membrane matrix. Atomic force microscopy (AFM) analysis revealed that the addition of N-CNTs leads to reduced surface roughness, suggesting a good dispersion of the N-CNTs in the matrix. Permeability studies revealed that the addition of N-CNTs to the polyethersulfone (PES) solution increased the water flux of the blend membrane by up to 70%. N-CNT loadings of 0.04 wt% in the blend membranes gave low surface contact angle of 55° and high fluxes. In addition, inclusion of N-CNTs enhanced the mechanical properties of the N-CNT/PES blend membranes. The use of N-CNTs in mixed matrix PES membranes is reported for the first time here. The result already suggests superior compatibility of the N-CNTs with PES compared to undoped CNTs, due to the high surface reactivity of the N-CNTs.

  5. Detection of Proteins on Blot Membranes

    PubMed Central

    Goldman, Aaron; Harper, Sandra; Speicher, David W.

    2017-01-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

  6. Unsupported palladium alloy membranes and methods of making same

    DOEpatents

    Way, J. Douglas; Thoen, Paul; Gade, Sabina K.

    2015-06-02

    The invention provides support-free palladium membranes and methods of making these membranes. Single-gas testing of the unsupported foils produced hydrogen permeabilities equivalent to thicker membranes produced by cold-rolling. Defect-free films as thin as 7.2 microns can be fabricated, with ideal H.sub.2/N.sub.2 selectivities as high as 40,000. Homogeneous membrane compositions may also be produced using these methods.

  7. Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

    PubMed

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

    1984-07-01

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

  8. Structure and properties of cellulose and cellulose/guar blend membranes prepared from the n-methyl morpholine n-oxide/water solvent system

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

    Luo, Mengkui; Winter, W.T.

    1995-12-01

    This paper describes membranes of cellulose or its blends with guar gums. Their morphology, hydration behavior, mechanical properties and permselectivity are all dependent upon preparation conditions. Wet membranes exhibit decreased strength but increased elasticity with increasing guar content. Morphologies of the wet membranes range from microporous to macrovoids to systems of regularly arranged conduits and could be formed in a reproducible manner. Dry membranes were invariably dense. Both wet and dry membranes had markedly higher permeation rates for molecules with 400 < M < 4000 than similarly treated commercial cellulose dialysis membranes and the rates increased with increasing guar content.more » Dried membranes of either cellulose or the blends showed appreciable permselectivity in this same intermediate molecular weight range which disappeared with increasing guar content.« less

  9. Electrospinning processed nanofibrous TiO2 membranes for photovoltaic applications

    NASA Astrophysics Data System (ADS)

    Onozuka, Katsuhiro; Ding, Bin; Tsuge, Yosuke; Naka, Takayuki; Yamazaki, Michiyo; Sugi, Shinichiro; Ohno, Shingo; Yoshikawa, Masato; Shiratori, Seimei

    2006-02-01

    We have recently fabricated dye-sensitized solar cells (DSSCs) comprising nanofibrous TiO2 membranes as electrode materials. A thin TiO2 film was pre-deposited on fluorine doped tin oxide (FTO) coated conducting glass substrate by immersion in TiF4 aqueous solution to reduce the electron back-transfer from FTO to the electrolyte. The composite polyvinyl acetate (PVac)/titania nanofibrous membranes can be deposited on the pre-deposited thin TiO2 film coated FTO by electrospinning of a mixture of PVac and titanium isopropoxide in N,N-dimethylformamide (DMF). The nanofibrous TiO2 membranes were obtained by calcining the electrospun composite nanofibres of PVac/titania as the precursor. Spectral sensitization of the nanofibrous TiO2 membranes was carried out with a ruthenium (II) complex, cis-dithiocyanate-N,N'-bis(2,2'-bipyridyl-4,4'-dicarboxylic acid) ruthenium (II) dihydrate. The results indicated that the photocurrent and conversion efficiency of electrodes can be increased with the addition of the pre-deposited TiO2 film and the adhesion treatment using DMF. Additionally, the dye loading, photocurrent, and efficiency of the electrodes were gradually increased by increasing the average thickness of the nanofibrous TiO2 membranes. The efficiency of the fibrous TiO2 photoelectrode with the average membrane thickness of 3.9 µm has a maximum value of 4.14%.

  10. Fabrication of Si3N4 nanowire membranes: free standing disordered nanopapers and aligned nanowire assemblies

    NASA Astrophysics Data System (ADS)

    Liu, Haitao; Fang, Minghao; Huang, Zhaohui; Huang, Juntong; Liu, Yan-gai; Wu, Xiaowen

    2016-08-01

    Herein, ultralong silicon nitride nanowires were synthesized via a chemical vapor deposition method by using the low-cost quartz and silicon powder as raw materials. Simple processes were used for the fabrication of disordered and ordered nanowire membranes of pure silicon nitride nanowires. The nanowires in the disordered nanopapers are intertwined with each other to form a paper-like structure which exhibit excellent flame retardancy and mechanical properties. Fourier-transform infrared spectroscopy and thermal gravity analysis were employed to characterize the refractory performance of the disordered nanopapers. Highly ordered nanowire membranes were also assembled through a three-phase assembly approach which make the Si3N4 nanowires have potential use in textured ceramics and semiconductor field. Moreover, the surface nanowires can also be modified to be hydrophobic; this characteristic make the as-prepared nanowires have the potential to be assembled by the more effective Langmuir-Blodgett method and also make the disordered nanopapers possess a super-hydrophobic surface.

  11. Detection of Proteins on Blot Membranes.

    PubMed

    Goldman, Aaron; Harper, Sandra; Speicher, David W

    2016-11-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  12. The Solid-Phase Synthesis of an Fe-N-C Electrocatalyst for High-Power Proton-Exchange Membrane Fuel Cells.

    PubMed

    Liu, Qingtao; Liu, Xiaofang; Zheng, Lirong; Shui, Jianglan

    2018-01-26

    The environmentally friendly synthesis of highly active Fe-N-C electrocatalysts for proton-exchange membrane fuel cells (PEMFCs) is desirable but remains challenging. A simple and scalable method is presented to fabricate Fe II -doped ZIF-8, which can be further pyrolyzed into Fe-N-C with 3 wt % of Fe exclusively in Fe-N 4 active moieties. Significantly, this Fe-N-C derived acidic PEMFC exhibits an unprecedented current density of 1.65 A cm -2 at 0.6 V and the highest power density of 1.14 W cm -2 compared with previously reported NPMCs. The excellent PEMFC performance can be attributed to the densely and atomically dispersed Fe-N 4 active moieties on the small and uniform catalyst nanoparticles. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Interaction of MreB-derived antimicrobial peptides with membranes.

    PubMed

    Saikia, Karabi; Chaudhary, Nitin

    2018-03-25

    Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor- and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts.

    PubMed Central

    Andrieu, N; Salvayre, R; Levade, T

    1994-01-01

    The hydrolysis of sphingomyelin (SPM) has been reported to mediate a number of responses to extracellular agents, including cytokines. The so-called SPM cycle may result from the activation of different types of sphingomyelinases (SPMases). We investigated the hypothetical contribution of acid lysosomal SPMase in the SPM signal-transduction pathway. We examined the ability of human skin fibroblasts with a genetic deficiency of acid lysosomal SPMase activity to respond to tumour necrosis factor alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). We report that both cytokines promoted SPM hydrolysis in fibroblasts derived from patients with Niemann-Pick disease or I-cell disease, similar to that observed in normal cells. Treatment of normal fibroblasts with cationic amphiphilic drugs resulted in inhibition of acid SPMase activity, but had no effect on cytokine-induced SPM turnover. In addition, TNF-alpha and IL-1 beta stimulated [3H]thymidine incorporation in Niemann-Pick fibroblasts, as in normal cells. Thus our results argue against a role for acid endolysosomal SPMase in mediating the cytokine-induced SPM signalling cascade. Images Figure 2 PMID:7980390

  15. Pressure modulation of Ras-membrane interactions and intervesicle transfer.

    PubMed

    Kapoor, Shobhna; Werkmüller, Alexander; Goody, Roger S; Waldmann, Herbert; Winter, Roland

    2013-04-24

    Proteins attached to the plasma membrane frequently encounter mechanical stresses, including high hydrostatic pressure (HHP) stress. Signaling pathways involving membrane-associated small GTPases (e.g., Ras) have been identified as critical loci for pressure perturbation. However, the impact of mechanical stimuli on biological outputs is still largely terra incognita. The present study explores the effect of HHP on the membrane association, dissociation, and intervesicle transfer process of N-Ras by using a FRET-based assay to obtain the kinetic parameters and volumetric properties along the reaction path of these processes. Notably, membrane association is fostered upon pressurization. Conversely, depending on the nature and lateral organization of the lipid membrane, acceleration or retardation is observed for the dissociation step. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provide first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kilobar (100 MPa) range.

  16. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    PubMed

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Calcium influx is required for endocytotic membrane retrieval

    PubMed Central

    Vogel, Steven S.; Smith, Robert M.; Baibakov, Boris; Ikebuchi, Yoshihide; Lambert, Nevin A.

    1999-01-01

    Cells use endocytotic membrane retrieval to compensate for excess surface membrane after exocytosis. Retrieval is thought to be calcium-dependent, but the source of this calcium is not known. We found that, in sea urchin eggs, endocytotic membrane retrieval required extracellular calcium. Inhibitors of P-type calcium channels—cadmium, ω-conotoxin MVIIC, ω-agatoxin IVA, and ω-agatoxin TK—blocked membrane retrieval; selective inhibitors of N-type and L-type channels did not. Treatment with calcium ionophores overcame agatoxin inhibition in a calcium-dependent manner. Cadmium blocked membrane retrieval when applied during the first 5 minutes after fertilization, the period when the membrane potential is depolarized. We conclude that calcium influx through ω-agatoxin-sensitive channels plays a key role in signaling for endocytotic membrane retrieval. PMID:10220411

  18. Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

    PubMed

    Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

    1992-05-15

    Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

  19. Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

    PubMed Central

    Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

    1992-01-01

    Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

  20. Study on surface adhesion of Plasma modified Polytetrafluoroethylene hollow fiber membrane

    NASA Astrophysics Data System (ADS)

    Chen, Jiangrong; Zhang, Huifeng; Liu, Guochang; Guo, Chungang; Lv, Jinglie; Zhangb, Yushan

    2018-01-01

    Polytetrafluoroethylene (PTFE) is popular membrane material because of its excellent thermal stability, chemical stability and mechanical stability. However, the low surface energy and non-sticky property of PTFE present challenges for modification. In the present study, plasma treatment was performed to improve the surface adhesion of PTFE hollow fiber membrane. The effect of discharge voltage, treatment time on the adhesion of PTFE hollow fiber membrane was symmetrically evaluated. Results showed that the plasma treatment method contributed to improve the surface activity and roughness of PTFE hollow fiber membrane, and the adhesion strength depend significantly on discharge voltage, which was beneficial to seepage pressure of PTFE hollow fiber membrane module. The adhesion strength of PTFE membrane by plasma treated at 220V for 3min reached as high as 86.2 N, far surpassing the adhesion strength 12.7 N of pristine membrane. Furthermore, improvement of content of free radical and composition analysis changes of the plasma modified PTFE membrane were investigated. The seepage pressure of PTFE membrane by plasma treated at 220V for 3min was 0.375 MPa, which means that the plasma treatment is an effective technique to improve the adhesion strength of membrane.

  1. An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.

    PubMed

    Pan, Yuan; Laird, Joseph G; Yamaguchi, David M; Baker, Sheila A

    2015-06-01

    Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane.

  2. An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors

    PubMed Central

    Pan, Yuan; Laird, Joseph G.; Yamaguchi, David M.; Baker, Sheila A.

    2015-01-01

    Purpose. Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. Methods. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. Results. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. Conclusions. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane. PMID:26030105

  3. Preparation of hierarchical structured nano-sized/porous poly(lactic acid) composite fibrous membranes for air filtration

    NASA Astrophysics Data System (ADS)

    Wang, Zhe; Pan, Zhijuan

    2015-11-01

    Hierarchical structured nano-sized/porous poly(lactic acid) (PLA-N/PLA-P) composite fibrous membranes with excellent air filtration performance were prepared via an electrospinning technique. Firstly, PLA-P fibers with different morphology were fabricated by varying the relative humidity, and the nanopores on fiber surface played a key role in improving the specific surface area and filtration performance of the resultant membranes. Secondly, hierarchical structure of PLA-N/PLA-P interlaced structured membranes and PLA-N/PLA-P double-layer structured membranes with different mass ratios for further enhanced air filtration performance were also successfully prepared by combining PLA-N fibers with PLA-P fibers. Filtration tests by measuring the penetration of sodium chloride (NaCl) aerosol particles with a 260 nm mass median diameter revealed that a moderate mass ratio of PLA-P fibers and PLA-N fibers contributed to improving the filtration performance of the hierarchical structured PLA-N/PLA-P composite membrane, and the double-layer structured PLA-N/PLA-P membrane possessed a higher filtration efficiency and quality factor than that of an interlaced structured PLA-N/PLA-P membrane with the same mass ratio. The as-prepared PLA-N/PLA-P double-layer structured membrane with a mass ratio of 1/5 showed a high filtration efficiency (99.999%) and a relatively low pressure drop (93.3 Pa) at the face velocity of 5.3 cm/s.

  4. Novel Inorganic/Polymer Composite Membranes for CO 2 Capture

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

    Ho, W.S. Winston; Dutta, Prabir K.; Schmit, Steve J.

    The objective of this project is to develop a cost-effective design and manufacturing process for new membrane modules that capture CO 2 from flue gas in coal-fired power plants. The membrane consisted of a thin selective layer including inorganic (zeolite) embedded in a polymer structure so that it can be made in a continuous manufacturing process. The membrane was incorporated in spiral-wound modules for the field test with actual flue gas at the National Carbon Capture Center (NCCC) in Wilsonville, AL and bench scale tests with simulated flue gas at the Ohio State University (OSU). Using the modules for post-combustionmore » CO 2 capture is expected to achieve the DOE target of $40/tonne CO 2 captured (in 2007 dollar) for 2025. Membranes with the amine-containing polymer cover layer on zeolite-Y (ZY) nanoparticles deposited on the polyethersulfone (PES) substrate were successfully synthesized. The membranes showed a high CO 2 permeance of about 1100 GPU (gas permeation unit, 1 GPU = 10 -6 cm 3 (STP)/(cm 2 • s • cm Hg), 3000 GPU = 10-6 mol/(m 2 • s • Pa)) with a high CO 2/N 2 selectivity of > 200 at the typical flue gas conditions at 57°C (about 17% water vapor in feed gas) and > 1400 GPU CO 2 permeance with > 500 CO 2/N 2 selectivity at 102°C (~ 80% water vapor). The synthesis of ZY nanoparticles was successfully scaled up, and the pilot-scale membranes were also successfully fabricated using the continuous membrane machine at OSU. The transport performance of the pilot-scale membranes agreed reasonably well with the lab-scale membranes. The results from both the lab-scale and scale-up membranes were used for the techno-economic analysis. The scale-up membranes were fabricated into prototype spiral-wound membrane modules for continuous testing with simulated or real flue gas. For real flue gas testing, we worked with NCCC, in consultation with TriSep Corporation, Gradient Technology and American Electric Power (AEP). The membrane module demonstrated

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  6. Changes in visceral adipose tissue plasma membrane lipid composition in old rats are associated with adipocyte hypertrophy with aging.

    PubMed

    Bonzón-Kulichenko, Elena; Moltó, Eduardo; Pintado, Cristina; Fernández, Alejandro; Arribas, Carmen; Schwudke, Dominik; Gallardo, Nilda; Shevchenko, Andrej; Andrés, Antonio

    2018-04-16

    Increased adiposity, through adipocyte hypertrophy and/or hyperplasia, characterizes aging and obesity. Both are leptin-resistant states, associated to disturbed lipid metabolism, reduced insulin sensitivity and inflammation. Nevertheless, fat tissue dysfunction appears earlier in obesity than in normal aging. In contrast, lipodystrophy is accompanied by diabetes, and improving the fat cell capacity to expand rescues the diabetic phenotype. Fat tissue dysfunction is extensively studied in the diet-induced obesity, but remains relatively neglected in the aging-associated obesity. In the Wistar rat, as occurs in humans, early or middle aging is accompanied by an increase in adiposity. Using this experimental model, we describe the molecular mechanisms contributing to the white adipose tissue (WAT) hypertrophy. WAT from middle-old age rats is characterized by decreased basal lipogenesis and lipolysis, increased esterification, as demonstrated by the higher TAG and cholesterol content in visceral WAT, and the maintenance of total ceramide levels within normal values. In addition, we describe alterations in the adipose tissue plasma membrane lipid composition, as increased total ether-phosphatidylcholine, sphingomyelin and free cholesterol levels that favor an enlarged fat cell size with aging. All these metabolic changes may be regarded as a survival advantage that prevents the aged rats from becoming overtly diabetic.

  7. Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation.

    PubMed

    Bae, Hyoung Eun; Gotfryd, Kamil; Thomas, Jennifer; Hussain, Hazrat; Ehsan, Muhammad; Go, Juyeon; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

    2015-07-06

    Detergents are an absolute requirement for studying the structure of membrane proteins. However, many conventional detergents fail to stabilise denaturation-sensitive membrane proteins, such as eukaryotic proteins and membrane protein complexes. New amphipathic agents with enhanced efficacy in stabilising membrane proteins will be helpful in overcoming the barriers to studying membrane protein structures. We have prepared a number of deoxycholate-based amphiphiles with carbohydrate head groups, designated deoxycholate-based glycosides (DCGs). These DCGs are the hydrophilic variants of previously reported deoxycholate-based N-oxides (DCAOs). Membrane proteins in these agents, particularly the branched diglucoside-bearing amphiphiles DCG-1 and DCG-2, displayed favourable behaviour compared to previously reported parent compounds (DCAOs) and conventional detergents (LDAO and DDM). Given their excellent properties, these agents should have significant potential for membrane protein studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B cells.

    PubMed

    Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

    2011-06-01

    Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by ~50-70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering.

  9. Omniphobic Membrane for Robust Membrane Distillation

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

    Lin, SH; Nejati, S; Boo, C

    2014-11-01

    In this work, we fabricate an omniphobic microporous membrane for membrane distillation (MD) by modifying a hydrophilic glass fiber membrane with silica nanoparticles followed by surface fluorination and polymer coating. The modified glass fiber membrane exhibits an anti-wetting property not only against water but also against low surface tension organic solvents that easily wet a hydrophobic polytetrafluoroethylene (PTFE) membrane that is commonly used in MD applications. By comparing the performance of the PTFE and omniphobic membranes in direct contact MD experiments in the presence of a surfactant (sodium dodecyl sulfate, SDS), we show that SDS wets the hydrophobic PTFE membranemore » but not the omniphobic membrane. Our results suggest that omniphobic membranes are critical for MD applications with feed waters containing surface active species, such as oil and gas produced water, to prevent membrane pore wetting.« less

  10. Erythrocyte membrane fatty acids in multiple myeloma patients.

    PubMed

    Jurczyszyn, Artur; Czepiel, Jacek; Gdula-Argasińska, Joanna; Czapkiewicz, Anna; Biesiada, Grażyna; Dróżdż, Mirosław; Perucki, William; Castillo, Jorge J

    2014-10-01

    Mounting data show that fatty acids (FA) and fatty acid synthase (FAS) function could be potential targets for multiple myeloma (MM) therapy. Our study aimed at comparing the FA composition of erythrocyte membranes of MM patients and healthy controls. MM patients had higher saturated FA and n-6 polyunsaturated FA (PUFA) and lower monounsaturated, n-3 PUFA and trans-FA indices than controls. The n-3/n-6 PUFA ratio was lower in MM patients and there was distinct clustering of variants of individual FA in MM patients. The FA content of erythrocyte membrane could serve as a diagnostic and/or predictive biomarker in MM. Copyright © 2014. Published by Elsevier Ltd.

  11. Amino-Functionalized Ceramic Capillary Membranes for Controlled Virus Retention.

    PubMed

    Bartels, Julia; Souza, Marina N; Schaper, Amelie; Árki, Pál; Kroll, Stephen; Rezwan, Kurosch

    2016-02-16

    A straightforward chemical functionalization strategy using aminosilanes for high-flux yttria-stabilized zirconia capillary membranes is presented (macroporous, d50 = 144 nm, open porosity =49%, membrane flux ∼150 L/(m(2)hbar)). Three different aminosilanes with one, two or three amino groups per silane molecule, namely 3-aminopropyltriethoxysilane (APTES), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AE-APTES) and N-(3-trimethoxysilylpropyl)diethylenetriamine (TPDA), are used to generate the amino-functionalized membranes. With a higher number of amino groups per silane molecule increased loading capacities between 0.44 and 1.01 accessible amino groups/nm(2) membrane are achieved. Streaming potential measurements confirm that the zeta-potential of the membrane surface is converted from negative (non-functionalized) to positive (amino-functionalized). By operation in dead-end filtration mode using the model virus MS2 (diameter = 25 nm, IEP = 3.9) the virus retention capacity of the amino-functionalized membranes is significantly increased and log reduction values (LRVs) of up to 9.6 ± 0.3 (TPDA) are obtained whereas a LRV < 0.3 is provided by the non-functionalized membranes. Long-term dead-end filtration experiments for 1 week reveal a high stability of immobilized aminosilanes (TPDA), being robust against leaching. By iterative backflushing with desorption buffer MS2-loaded membranes are successfully regenerated being reusable for a new filtration cycle. The presented functionalization platform is highly promising for controlled virus retention.

  12. 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

  13. Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization

    PubMed Central

    Wu, Tingting; Shi, Zheng; Baumgart, Tobias

    2014-01-01

    The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease. PMID:24755653

  14. Biopores/membrane proteins in synthetic polymer membranes.

    PubMed

    Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

    2017-04-01

    Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

  15. Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography.

    PubMed

    Liberton, Michelle; Austin, Jotham R; Berg, R Howard; Pakrasi, Himadri B

    2011-04-01

    Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.

  16. Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models.

    PubMed Central

    Webb, M. S.; Harasym, T. O.; Masin, D.; Bally, M. B.; Mayer, L. D.

    1995-01-01

    This study reports on the development of a liposomal formulation of vincristine with significantly enhanced stability and biological properties. The in vitro and in vivo pharmacokinetic, tumour delivery and efficacy properties of liposomal vincristine formulations based on sphingomyelin (SM) and cholesterol were compared with liposomes composed of distearoylphosphatidylcholine (DSPC) and cholesterol. SM/cholesterol liposomes had significantly greater in vitro stability than did similar DSPC/cholesterol liposomes. SM/cholesterol liposomes also had significantly improved biological properties compared with DSPC/cholesterol. Specifically, SM/cholesterol liposomes administered intravenously retained 25% of the entrapped vincristine after 72 h in the circulation, compared with 5% retention in DSPC/cholesterol liposomes. The improved retention properties of SM/cholesterol liposomes resulted in plasma vincristine levels 7-fold higher than in DSPC/cholesterol liposomes. The improved circulation lifetime of vincristine in SM/cholesterol liposomes correlated with increased vincristine accumulation in peritoneal ascitic murine P388 tumours and in subcutaneous solid A431 human xenograft tumours. Increased vincristine delivery to tumours was also accompanied by increased anti-tumour efficacy. Treatment with SM/cholesterol liposomal formulations of vincristine resulted in greater than 50% cures in mice bearing ascitic P388 tumours, an activity that could not be achieved with the DSPC/cholesterol formulation. Similarly, treatment of mice with severe combined immunodeficiency (SCID) bearing solid human A431 xenograft tumours with SM/cholesterol vincristine formulations delayed the time required for 100% increase in tumour mass to > 40 days, compared with 5 days, 7 days and 14 days for mice receiving no treatment or treatment with free vincristine or DSPC/cholesterol formulations of vincristine respectively. PMID:7547237

  17. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

    PubMed

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Membrane Disordering by Eicosapentaenoic Acid in B Lymphomas Is Reduced by Elongation to Docosapentaenoic Acid as Revealed with Solid-State Nuclear Magnetic Resonance Spectroscopy of Model Membranes.

    PubMed

    Harris, Mitchell; Kinnun, Jacob J; Kosaraju, Rasagna; Leng, Xiaoling; Wassall, Stephen R; Shaikh, Saame Raza

    2016-07-01

    Plasma membrane organization is a mechanistic target of n-3 (ω-3) polyunsaturated fatty acids. Previous studies show that eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) differentially disrupt plasma membrane molecular order to enhance the frequency and function of B lymphocytes. However, it is not known whether EPA and DHA affect the plasma membrane organization of B lymphomas differently to influence their function. We tested whether EPA and DHA had different effects on membrane order in B lymphomas and liposomes and studied their effects on B-lymphoma growth. B lymphomas were treated with 25 μmol EPA, DHA, or serum albumin control/L for 24 h. Membrane order was measured with fluorescence polarization, and cellular fatty acids (FAs) were analyzed with GC. Growth was quantified with a viability assay. (2)H nuclear magnetic resonance (NMR) studies were conducted on deuterated phospholipid bilayers. Treating Raji, Ramos, and RPMI lymphomas for 24 h with 25 μmol EPA or DHA/L lowered plasma membrane order by 10-40% relative to the control. There were no differences between EPA and DHA on membrane order for the 3 cell lines. FA analyses revealed complex changes in response to EPA or DHA treatment and a large fraction of EPA was converted to docosapentaenoic acid (DPA; 22:5n-3). NMR studies, which were used to understand why EPA and DHA had similiar membrane effects, showed that phospholipids containing DPA, similar to DHA, were more ordered than those containing EPA. Finally, treating B lymphomas with 25 μmol EPA or DHA/L did not increase the frequency of B lymphomas compared with controls. The results establish that 25 μmol EPA and DHA/L equally disrupt membrane order and do not promote B lymphoma growth. The data open a new area of investigation, which is how EPA's conversion to DPA substantially moderates its influence on membrane properties. © 2016 American Society for Nutrition.

  19. Carbon dioxide (hydrogen sulfide) membrane separations and WGS membrane reactor modeling for fuel cells

    NASA Astrophysics Data System (ADS)

    Huang, Jin

    Acid-gas removal is of great importance in many environmental or energy-related processes. Compared to current commercial technologies, membrane-based CO2 and H2S capture has the advantages of low energy consumption, low weight and space requirement, simplicity of installation/operation, and high process flexibility. However, the large-scale application of the membrane separation technology is limited by the relatively low transport properties. In this study, CO2 (H2S)-selective polymeric membranes with high permeability and high selectivity have been studied based on the facilitated transport mechanism. The membrane showed facilitated effect for both CO2 and H2S. A CO2 permeability of above 2000 Barrers, a CO2/H2 selectivity of greater than 40, and a CO2/N2 selectivity of greater than 200 at 100--150°C were observed. As a result of higher reaction rate and smaller diffusing compound, the H2S permeability and H2S/H2 selectivity were about three times higher than those properties for CO2. The novel CO2-selective membrane has been applied to capture CO 2 from flue gas and natural gas. In the CO2 capture experiments from a gas mixture with N2 and H2, a permeate CO 2 dry concentration of greater than 98% was obtained by using steam as the sweep gas. In CO2/CH4 separation, decent CO 2 transport properties were obtained with a feed pressure up to 500 psia. With the thin-film composite membrane structure, significant increase on the CO2 flux was achieved with the decrease of the selective layer thickness. With the continuous removal of CO2, CO2-selective water-gas-shift (WGS) membrane reactor is a promising approach to enhance CO conversion and increase the purity of H2 at process pressure under relatively low temperature. The simultaneous reaction and transport process in the countercurrent WGS membrane reactor was simulated by using a one-dimensional non-isothermal model. The modeling results show that a CO concentration of less than 10 ppm and a H2 recovery of greater

  20. Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts [High-Resolution Chemical Imaging of Sphingolipid Distribution in the Plasma Membrane

    DOE PAGES

    Frisz, Jessica F.; Lou, Kaiyan; Klitzing, Haley A.; ...

    2013-01-28

    Sphingolipids play important roles in plasma membrane structure and cell signaling. Yet, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of 15N-enriched ions from metabolically labeled 15N-sphingolipids in the plasma membrane using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids$-$both in living cells and during fixation of living cells$-$exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15Nsphingolipid microdomains with mean diametersmore » of ~200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts, and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.« less

  1. Bacterial nanocellulose/Nafion composite membranes for low temperature polymer electrolyte fuel cells

    NASA Astrophysics Data System (ADS)

    Jiang, Gao-peng; Zhang, Jing; Qiao, Jin-li; Jiang, Yong-ming; Zarrin, Hadis; Chen, Zhongwei; Hong, Feng

    2015-01-01

    Novel nanocomposite membranes aimed for both proton-exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) are presented in this work. The membranes are based on blending bacterial nanocellulose pulp and Nafion (abbreviated as BxNy, where x and y indicates the mass ratio of bacterial cellulose to Nafion). The structure and properties of BxNy membranes are characterized by FTIR, SEM, TG, DMA and EIS, along with water uptake, swelling behavior and methanol permeability tests. It is found that the BxNy composite membranes with reinforced concrete-like structure show excellent mechanical and thermal stability regardless of annealing. The water uptake plus area and volume swelling ratios are all decreased compared to Nafion membranes. The proton conductivities of pristine and annealed B1N9 are 0.071 and 0.056 S cm-1, respectively, at 30 °C and 100% humidity. Specifically, annealed B1N1 exhibited the lowest methanol permeability of 7.21 × 10-7 cm2 s-1. Through the selectivity analysis, pristine and annealed B1N7 are selected to assemble the MEAs. The performances of annealed B1N7 in PEMFC and DMFC show the maximum power densities of 106 and 3.2 mW cm-2, respectively, which are much higher than those of pristine B1N7 at 25 °C. The performances of the pristine and annealed B1N7 reach a level as high as 21.1 and 20.4 mW cm-2 at 80 °C in DMFC, respectively.

  2. A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

    PubMed

    Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

    2006-01-24

    Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

  3. Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

    PubMed Central

    Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

    2009-01-01

    Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

  4. Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

    PubMed

    Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

    2008-09-01

    The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

  5. Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

    PubMed

    de Oliveira, Guilherme A P; Pereira, Elen G; Ferretti, Giulia D S; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

    2013-09-27

    c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

  6. Anionic membrane and ionomer based on poly(2,6-dimethyl-1,4-phenylene oxide) for alkaline membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Ong, Ai Lien; Saad, Saeed; Lan, Rong; Goodfellow, Robert J.; Tao, Shanwen

    2011-10-01

    Hydroxyl-ion conductive poly(2,6-dimethyl-1,4-phenylene oxide) (PPO) membranes with different characteristics were prepared via relatively simple bromination/amination serial reactions with reduced number of involved chemicals and shorter reaction time. The effects of reactants ratio, reaction atmosphere, polymer concentration, casting solvent, and hydroxylation treatment on reaction were investigated in details. The microstructure, water uptake, swelling ratio, ion-exchange capacity and ionic conductivity of the membranes were also studied. The obtained results demonstrate that, the ionic conductivity of the membrane is dependent on casting solvent. The N-methyl-2-pyrrolidonecast membrane exhibits the highest conductivity with the thinnest film. Although the membrane was prepared via a relatively simple preparation route with least toxic chemicals, a competitive ionic conductivity value of 1.64 × 10-2 S cm-1 was achieved at 60 °C. A power density of 19.5 mW cm-2 has been demonstrated from the alkaline membrane fuel cell operated at 70 °C, assembled from the entirely homemade membrane electrode assembly without any hot-pressing.

  7. Membranes, methods of making membranes, and methods of separating gases using membranes

    DOEpatents

    Ho, W. S. Winston

    2012-10-02

    Membranes, methods of making membranes, and methods of separating gases using membranes are provided. The membranes can include at least one hydrophilic polymer, at least one cross-linking agent, at least one base, and at least one amino compound. The methods of separating gases using membranes can include contacting a gas stream containing at least one of CO.sub.2, H.sub.2S, and HCl with one side of a nonporous and at least one of CO.sub.2, H.sub.2S, and HCl selectively permeable membrane such that at least one of CO.sub.2, H.sub.2S, and HCl is selectively transported through the membrane.

  8. Antifouling enhancement of polyimide membrane by grafting DEDA-PS zwitterions.

    PubMed

    Zhang, Dong Yan; Xiong, Shu; Shi, Yu Sheng; Zhu, Jun; Hu, Qiao Li; Liu, Jie; Wang, Yan

    2018-05-01

    In order to improve the water flux and antifouling property of polyimide (PI) membrane, zwitterions are grafted on PI membrane surface via a two-step modification route by reactions with N,N-diethylethylenediamine (DEDA) and 1,3-propane sultone (PS) sequentially. The reaction mechanism and physicochemical properties of membranes are confirmed via various characterization techniques. The anti-biofouling performance of the zwitterion-grafted PI membranes is evaluated by bacterial suspension immersion tests in Escherichia coli (E. coli) and staphylococcus aureus (S. aureus) solutions. The antifouling property is assessed via the filtration test using the bovine serum albumin (BSA) and dodecyl trimethyl ammonium bromide (DTAB) aqueous feed solutions. The effect of the reaction time with DEDA in the zwitterion-grafted process on the antifouling property is further investigated systematically. The results show that both the anti-biofouling and antifouling performances of zwitterion-grafted PI membranes are significantly improved. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

    PubMed Central

    Paslawski, Wojciech; Lillelund, Ove K.; Kristensen, Julie Veje; Schafer, Nicholas P.; Baker, Rosanna P.; Urban, Sinisa; Otzen, Daniel E.

    2015-01-01

    Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated. PMID:26056273

  10. A Feasibility Study of Ammonia Recovery from Coking Wastewater by Coupled Operation of a Membrane Contactor and Membrane Distillation

    PubMed Central

    Horng, Ren-Yang; Hsu, Shu-Fang; Chen, Shiao-Shing; Ho, Chia-Hua

    2018-01-01

    More than 80% of ammonia (NH3) in the steel manufacturing process wastewater is contributed from the coking wastewater, which is usually treated by biological processes. However, the NH3 in the coking wastewater is typically too high for biological treatment due to its inhibitory concentration. Therefore, a two-stage process including a hollow fiber membrane contactor (HFMC) and a modified membrane distillation (MD) system was developed and applied to reduce and recover NH3 from coking wastewater. The objectives of this paper are to evaluate different membrane materials, receiving solutions, and operation parameters for the system, remove NH3 from the coking wastewater to less than 300 mg N/L, which is amenable to the biological process, and recover ammonia solution for reuse. As a result, the polytetrafluoroethylene (PTFE) HFMC using sulfuric acid as a receiving solution can achieve a maximum NH3-N transmembrane flux of 1.67 g N/m2·h at pH of 11.5 and reduce NH3 in the coking wastewater to less than 300 mg N/L. The NH3 in the converted ammonium sulfate ((NH4)2SO4) was then recovered by the modified MD using ice water as the receiving solution to produce ≥3% of ammonia solution for reuse. PMID:29510505

  11. Changes in Sinus Membrane Thickness After Lateral Sinus Floor Elevation: A Radiographic Study.

    PubMed

    Makary, Christian; Rebaudi, Alberto; Menhall, Abdallah; Naaman, Nada

    2016-01-01

    To radiographically monitor sinus membrane swelling after lateral sinus floor elevation surgery at short and long healing periods. For 26 patients seeking posterior maxillary implant-supported reconstruction, 32 lateral sinus floor elevations were performed using Piezosurgery. Sinus membranes were grafted using synthetic calcium phosphate bone substitutes, and graft volume was measured in cubic centimeters for each case. Cone beam computed tomography (CBCT) examination was conducted preoperatively in all patients and for each grafted sinus at 1 day (n = 8), 2 days (n = 9), 3 days (n = 8), or 7 days (n = 7) after surgery. Control CBCT was then performed for all patients at 3, 6, and 12 months after surgery. Sinus membrane thickness was measured on cross-sectional CBCT images at nine standardized points per sinus, before lateral sinus floor elevation and at all postoperative examinations. Mean sinus membrane thickness was 0.73 mm before surgery, and 5 mm, 4.1 mm, 5.9 mm, and 7 mm, respectively, at 1, 2, 3, and 7 days after surgery. First week combined postoperative CBCT measurements of membrane thickness was 5.4 mm, then 1.3, 0.68, and 0.39 mm at 3, 6, and 12 months, respectively, after surgery. Membrane thickness significantly increased the first week after surgery and gradually decreased significantly at 3, 6, and 12 months in all groups (P < .001). First-week postoperative measurements showed a significant increase in membrane thickness at 3 days compared with the 1- and 2-day results (P < .001) and at 7 days compared with all other time points (P < .001). Membrane thickness at 2 days did not change significantly compared with 1-day measurements. Larger graft volume was positively correlated with an increase in membrane thickness after surgery at all time points (n = 32; r = 0.527; P < .001). After lateral sinus floor elevation surgery, transient swelling of sinus membrane is observed. It reaches a peak value 7 days after surgery and completely resolves over

  12. Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

    PubMed

    Ho, Hoang-Yen; Moffat, Ryan C; Patel, Rupal V; Awah, Franklin N; Baloue, Kaitrin; Crowe, David L

    2010-09-01

    Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  13. Membrane Raft Organization Is More Sensitive to Disruption by (n-3) PUFA Than Nonraft Organization in EL4 and B Cells123

    PubMed Central

    Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

    2011-01-01

    Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate by ~50–70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering. PMID:21525263

  14. Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair.

    PubMed

    Amado, Sandra; Rodrigues, Jorge M; Luís, Ana L; Armada-da-Silva, Paulo A S; Vieira, Márcia; Gartner, Andrea; Simões, Maria J; Veloso, António P; Fornaro, Michele; Raimondo, Stefania; Varejão, Artur S P; Geuna, Stefano; Maurício, Ana C

    2010-02-11

    Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat.

  15. Sphingolipid Metabolism Correlates with Cerebrospinal Fluid Beta Amyloid Levels in Alzheimer’s Disease

    PubMed Central

    Fonteh, Alfred N.; Ormseth, Cora; Chiang, Jiarong; Cipolla, Matthew; Arakaki, Xianghong; Harrington, Michael G.

    2015-01-01

    Sphingolipids are important in many brain functions but their role in Alzheimer’s disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired

  16. Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes.

    PubMed

    Kumazawa, T; Nomura, T; Kurihara, K

    1988-02-23

    Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.

  17. The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

    PubMed Central

    Varela Chavez, Carolina; Haustant, Georges Michel; Baron, Bruno; England, Patrick; Chenal, Alexandre; Pauillac, Serge; Blondel, Arnaud; Popoff, Michel-Robert

    2016-01-01

    Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. PMID:27023605

  18. Cleaning Aged EPDM Rubber Roofing Membrane Material for Patching: Laboratory Investigations and Recommendations

    DTIC Science & Technology

    1992-08-01

    Cleaning Aged EPDM Rubber Roofing Membrane Material for Patching: Laboratory Investigations and Recommendations Walter J. Rossiter, Jr. T N n-’T ic...condition of the aged EPDM rubber before bonding. This study assessed the effectiveness of different cleaning methods for preparing aged EPDM membranes for...REPORT DATE 3. REPORT TYPE AND DATES COVERED August 1992 Final 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Cleaning Aged EPDM Rubber Roofing Membrane

  19. Desolventizing of Jatropha curcas oil from azeotropes of solvents using ceramic membranes.

    PubMed

    Carniel, Naira; Zabot, Giovani L; Paliga, Marshall; Mignoni, Marcelo L; Mazutti, Marcio A; Priamo, Wagner L; Oliveira, J V; Di Luccio, Marco; Tres, Marcus V

    2017-12-01

    The separation of Jatropha curcas oil from azeotropes of ethyl alcohol-n-hexane and isopropyl alcohol-n-hexane using ceramic membranes with different cutoffs (5, 10 and 20 kDa) is presented. The mass ratios of oil:azeotropes (O:S) studied were 1:3 for feeding pressures of 0.1, 0.2 and 0.3 MPa, and 1:1 for the feeding pressure of 0.1 MPa. Isopropyl alcohol was the best solvent for the membranes conditioning to permeate n-hexane (240 kg/m 2  h). In the separation of J. curcas oil and azeotropes of solvents, both membranes showed oil retention and total flux decreases with time. Overall, the lowest decrease in the retentions was reached in the 5 kDa membrane, while the lowest decrease in the total flux was reached in the 20 kDa. In the separation of oil and ethyl alcohol-n-hexane azeotrope, the best retention at 60 min of the process was equal to 17.3 wt% in the 20 kDa membrane at 0.3 MPa and O:S ratio equalled to 1:3. In this condition, the total permeate flux was 17.5 kg/m 2  h. Different retentions and permeabilities are provided when changing the O:S ratio, the feeding pressure and the molecular weight cutoff of membranes.

  20. Influence of diatomite addition on membrane fouling and performance in a submerged membrane bioreactor.

    PubMed

    Yang, Xiao-Li; Song, Hai-Liang; Lu, Ji-Lai; Fu, Da-Fang; Cheng, Bing

    2010-12-01

    This paper examined the effect of diatomite addition on membrane fouling and process performance in an anoxic/oxic submerged membrane bioreactor (A/O MBR). Particle size distribution, molecular weight distribution and microbial activity have been investigated to characterize the sludge mixed liquor. Results show that diatomite addition is a reliable and effective approach in terms of both membrane fouling mitigation and pollutants removal improvement. The MBR system with diatomite addition of 50 mg/L enhanced the removal of COD, TN and TP by 0.9%, 6.9% and 31.2%, respectively, as compared to the control MBR (without diatomite addition). The NH(4)-N removal always maintained at a high level of over 98% irrespective of diatomite addition. Due to the hybrid effect of adsorption and co-precipitation on fine colloids and dissolved organic matter (DOM) from the addition of diatomite, a reduction in foulants amount, an increase in microbial floc size and an improvement in sludge settleability have been achieved simultaneously. As a result, the membrane fouling rate was mitigated successfully. 2010 Elsevier Ltd. All rights reserved.

  1. Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

    PubMed Central

    Lehto, M T; Sharom, F J

    1998-01-01

    Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion

  2. [Effect of Membrane Wettability on Membrane Fouling and Chemical Durability of SPG Membranes].

    PubMed

    Zhang, Jing; Xiao, Tai-min; Zhang, Jing; Cao, Li-ya; Du, Ya-wei; Liu, Chun; Zhang, Lei

    2015-05-01

    Shirasu porous glass (SPG) membranes have been applied for microbubble aeration in aerobic wastewater treatment. In the present study, both hydrophilic and hydrophobic SPG membranes were used in a microbubble-aerated biofilm reactor with online chemical cleaning, and their membrane fouling and chemical durability were determined to be strongly dependent on the membrane wettability. The fouling layer formed on the surface of both membranes was confirmed to be mainly organic fouling, and the hydrophobic membrane showed a relatively stronger resistance to the organic fouling. The severe chemical corrosion of the hydrophilic membrane was observed due to exposure to the alkaline sodium hypochlorite solution used for chemical cleaning, which resulted in significant increases in the median pore diameter and the porosity. On the other hand, the pore structure of the hydrophobic membrane changed slightly when exposed to the alkaline sodium hypochlorite solution, suggesting its strong alkali-resistance due to the non-wetting surface. However, the surface hydrophobic groups of hydrophobic membrane could be oxidized by sodium hypochlorite solution, resulting in more wettable membrane surface. The hydrophobic membrane also showed better performance in the respects of oxygen transfer, contaminant removal and energy-saving. Therefore, the hydrophobic membrane seemed more appropriate to be applied for microbubble aeration in aerobic wastewater treatment process.

  3. A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

    PubMed

    Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

    2016-01-01

    The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

  4. Palladium-Zeolite nanofiber as an effective recyclable catalyst membrane for water treatment.

    PubMed

    Choi, Jungsu; Chan, Sophia; Yip, Garriott; Joo, Hyunjong; Yang, Heejae; Ko, Frank K

    2016-09-15

    Zeolite is an exciting natural material due to its unique capability of ammonium nitrogen (NH3N) adsorption in water. In this study, multifunctional hybrid composites of zeolite/palladium (Ze/Pd) on polymer nanofiber membranes were fabricated and explored for sustainable contaminant removal. SEM and XRD demonstrated that zeolite and palladium nanoparticles were uniformly distributed and deposited on the nanofibers. NH3N recovery rate was increased from 23 to 92% when palladium coated zeolite was embedded on the nanofiber. Multifunctional nanofibers of Ze/Pd membranes were able to adsorb NH3N on the zeolites placed on the surface of fibers and palladium catalysts were capable of selective oxidation of NH3N to N2 gas. The cycling of NH3N adsorption-oxidation, high flux, hydrophilicity, and flexibility of the membrane makes it a strong candidate for water treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes.

    PubMed

    Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo

    2013-06-01

    Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

  6. Separation of methane-nitrogen mixtures using synthesis vertically aligned carbon nanotube membranes

    NASA Astrophysics Data System (ADS)

    Gilani, Neda; Daryan, Jafar Towfighi; Rashidi, Alimorad; Omidkhah, Mohammad Reza

    2012-03-01

    In this paper, capabilities of carbon nanotube (CNT) membranes fabricated in cylindrical pores of anodic aluminum oxide (AAO) substrate to separate the binary mixtures of CH4/N2 are studied experimentally. For this purpose, the permeability and selectivity of three CNT/AAO membranes with different growth time as 6 h, 12 h and 18 h are investigated. CNTs are grown vertically through holes of AAO with average pore diameter of 45 nm by chemical vapor deposition (CVD) of acetylene gas. CNT/AAO membranes with the same CNTs' outer diameters and different inner diameters are synthesized. The AAO are characterized by SEM analysis. In addition, SEM, TEM, BET N2 adsorption analysis and Raman spectroscopy are employed to characterize aligned CNTs. Study on permeability and selectivity of membranes for three binary mixtures of CH4/N2 showed that when the CNT inner diameters are 34 nm and 24 nm, viscous flow is the governing mechanism and insignificant selectivities of 1.2-1.24 are achieved. However, the membrane with CNT inner diameter and wall thickness of 8 nm and 16 nm respectively is considerably selective for CH4 over N2. It was also found that CH4 mole fraction in the feed and upstream feed pressure have major effect on permeability and selectivity. The membrane with 18 h synthesis time showed the selectivity is in the range of 1.8-3.85. The enhancement factor for N2 single gas diffusivity was also found to be about three times larger than that predicted by Knudsen diffusion model.

  7. Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

    PubMed Central

    West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

    2005-01-01

    Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

  8. Intramolecular Dynamics within the N-Cap-SH3-SH2 Regulatory Unit of the c-Abl Tyrosine Kinase Reveal Targeting to the Cellular Membrane*♦

    PubMed Central

    de Oliveira, Guilherme A. P.; Pereira, Elen G.; Ferretti, Giulia D. S.; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L.

    2013-01-01

    c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl+ cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target. PMID:23928308

  9. High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice

    PubMed Central

    Kahle, M.; Schäfer, A.; Seelig, A.; Schultheiß, J.; Wu, M.; Aichler, M.; Leonhardt, J.; Rathkolb, B.; Rozman, J.; Sarioglu, H.; Hauck, S.M.; Ueffing, M.; Wolf, E.; Kastenmueller, G.; Adamski, J.; Walch, A.; Hrabé de Angelis, M.; Neschen, S.

    2014-01-01

    Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid

  10. Mimicking the cell membrane: bio-inspired simultaneous functions with monovalent anion selectivity and antifouling properties of anion exchange membrane

    PubMed Central

    Zhao, Yan; Liu, Huimin; Tang, Kaini; Jin, Yali; Pan, Jiefeng; der Bruggen, Bart Van; Shen, Jiangnan; Gao, Congjie

    2016-01-01

    A new bio-inspired method was applied in this study to simultaneously improve the monovalent anion selectivity and antifouling properties of anion exchange membranes (AEMs). Three-layer architecture was developed by deposition of polydopamine (PDA) and electro-deposition of N-O-sulfonic acid benzyl chitosan (NSBC). The innermost and outermost layers were PDA with different deposition time. The middle layer was prepared by NSBC. Fourier transform infrared spectroscopy and scanning electron microscopy confirmed that PDA and NSBC were successfully modified on the surfaces of AEMs. The contact angle of the membranes indicated an improved hydrophilicity of the modified membranes. A series of electrodialysis experiments in which Cl−/SO42− separation was studied, demonstrating the monovalent anion selectivity of the samples. The Cl−/SO42− permselectivity of the modified membranes can reach up to 2.20, higher than that of the commercial membrane (only 0.78) during 90 minutes in electrodialysis (ED). The increase value of the resistance of the membranes was also measured to evaluate the antifouling properties. Sodium dodecyl benzene sulfonate (SDBS) was used as the fouling material in the ED process and the membrane area resistance of modified membrane increase value of was only 0.08 Ωcm2 30 minutes later. PMID:27853255

  11. Mimicking the cell membrane: bio-inspired simultaneous functions with monovalent anion selectivity and antifouling properties of anion exchange membrane.

    PubMed

    Zhao, Yan; Liu, Huimin; Tang, Kaini; Jin, Yali; Pan, Jiefeng; der Bruggen, Bart Van; Shen, Jiangnan; Gao, Congjie

    2016-11-17

    A new bio-inspired method was applied in this study to simultaneously improve the monovalent anion selectivity and antifouling properties of anion exchange membranes (AEMs). Three-layer architecture was developed by deposition of polydopamine (PDA) and electro-deposition of N-O-sulfonic acid benzyl chitosan (NSBC). The innermost and outermost layers were PDA with different deposition time. The middle layer was prepared by NSBC. Fourier transform infrared spectroscopy and scanning electron microscopy confirmed that PDA and NSBC were successfully modified on the surfaces of AEMs. The contact angle of the membranes indicated an improved hydrophilicity of the modified membranes. A series of electrodialysis experiments in which Cl - /SO 4 2- separation was studied, demonstrating the monovalent anion selectivity of the samples. The Cl - /SO 4 2- permselectivity of the modified membranes can reach up to 2.20, higher than that of the commercial membrane (only 0.78) during 90 minutes in electrodialysis (ED). The increase value of the resistance of the membranes was also measured to evaluate the antifouling properties. Sodium dodecyl benzene sulfonate (SDBS) was used as the fouling material in the ED process and the membrane area resistance of modified membrane increase value of was only 0.08 Ωcm 2 30 minutes later.

  12. Mimicking the cell membrane: bio-inspired simultaneous functions with monovalent anion selectivity and antifouling properties of anion exchange membrane

    NASA Astrophysics Data System (ADS)

    Zhao, Yan; Liu, Huimin; Tang, Kaini; Jin, Yali; Pan, Jiefeng; der Bruggen, Bart Van; Shen, Jiangnan; Gao, Congjie

    2016-11-01

    A new bio-inspired method was applied in this study to simultaneously improve the monovalent anion selectivity and antifouling properties of anion exchange membranes (AEMs). Three-layer architecture was developed by deposition of polydopamine (PDA) and electro-deposition of N-O-sulfonic acid benzyl chitosan (NSBC). The innermost and outermost layers were PDA with different deposition time. The middle layer was prepared by NSBC. Fourier transform infrared spectroscopy and scanning electron microscopy confirmed that PDA and NSBC were successfully modified on the surfaces of AEMs. The contact angle of the membranes indicated an improved hydrophilicity of the modified membranes. A series of electrodialysis experiments in which Cl-/SO42- separation was studied, demonstrating the monovalent anion selectivity of the samples. The Cl-/SO42- permselectivity of the modified membranes can reach up to 2.20, higher than that of the commercial membrane (only 0.78) during 90 minutes in electrodialysis (ED). The increase value of the resistance of the membranes was also measured to evaluate the antifouling properties. Sodium dodecyl benzene sulfonate (SDBS) was used as the fouling material in the ED process and the membrane area resistance of modified membrane increase value of was only 0.08 Ωcm2 30 minutes later.

  13. Development of membranes and a study of their interfaces for rechargeable lithium-air battery

    NASA Astrophysics Data System (ADS)

    Kumar, Jitendra; Kumar, Binod

    This paper describes an investigation with an objective to screen and select high performance membrane materials for a working, rechargeable lithium-air battery. Membrane laminates comprising glass-ceramic (GC) and polymer-ceramic (PC) membranes were assembled, evaluated and analyzed. A superionic conducting GC membrane with a chemical composition of Li 1+ xAl xGe 2- x(PO 4) 3 (x = 0.5) was used. Polymer membranes comprising of PC(BN), PC(AlN), PC(Si 3N 4) and PC(Li 2O) electrochemically coupled the GC membrane with the lithium anode. The cell and membrane laminates were characterized by determining cell conductivity, open circuit voltage and carrier concentration and its mobility. The measurements identified Li 2O and BN as suitable dopants in polymer matrix which catalyzed anodic charge transfer reaction, formed stable SEI layer and provided high lithium ion conductivity.

  14. Pervaporation separation of thiophene-heptane mixtures with polydimethylsiloxane (PDMS) membrane for desulfurization.

    PubMed

    Chen, Jian; Li, Jiding; Qi, Rongbin; Ye, Hong; Chen, Cuixian

    2010-01-01

    Cross-linked polydimethylsiloxane (PDMS)-polyetherimide (PEI) composite membranes were prepared, in which asymmetric microporous PEI membrane prepared with phase inversion method was acted as the microporous supporting layer in the flat-plate composite membrane. Membrane characterization was conducted by Fourier transform infrared and scanning electronic microscopy analysis. The composite membranes were employed in pervaporation separation of n-heptane-thiophene mixtures. Effect of amount of PDMS, cross-linking temperature, amount of cross-linking agent, and cross-linking time on the separation efficiency of n-heptane-thiophene mixtures was investigated experimentally. Experiment results demonstrated that 80-100 degrees degrees C of cross-linking temperature was more preferable for practical application, as the amount of cross-linking agent was up to 20 wt.%, and 25 wt.% of PDMS amount was more optimal as far as flux and sulfur enrichment factor were concerned. In addition, the swelling degree of and stableness of composite membrane during long-time operation were studied, which should be significant for practical application.

  15. Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-Releasing Nanocellulose-Chitosan Packaging Membranes.

    PubMed

    Sundaram, Jaya; Pant, Jitendra; Goudie, Marcus J; Mani, Sudhagar; Handa, Hitesh

    2016-06-29

    Biodegradable composite membranes with antimicrobial properties consisting of nanocellulose fibrils (CNFs), chitosan, and S-nitroso-N-acetyl-d-penicillamine (SNAP) were developed and tested for food packaging applications. As a nitric oxide donor, SNAP was encapsulated into completely dispersed chitosan in 100 mL of 0.1 N acetic acid and was thoroughly mixed with CNFs to produce a composite membrane. The fabricated membranes had a uniform dispersion of chitosan and SNAP within the CNFs, which was confirmed through scanning electron microscopy (SEM) micrographs and a chemiluminescence nitric oxide analyzer. The membranes prepared without SNAP showed lower water vapor permeability than that of the membranes with SNAP. The addition of SNAP resulted in a decrease in Young's modulus for both two- and three-layer membrane configurations. Antimicrobial property evaluation of SNAP-incorporated membranes showed an effective zone of inhibition against bacterial strains of Enterococcus faecalis, Staphylococcus aureus, and Listeria monocytogenes and demonstrated its potential applications for food packaging.

  16. Membrane water-flow rate in electrolyzer cells with a solid polymer electrolyte (SPE)

    NASA Astrophysics Data System (ADS)

    Li, Xiaojin; Qu, Shuguo; Yu, Hongmei; Hou, Ming; Shao, Zhigang; Yi, Baolian

    Water-flow rate across Nafion membrane in SPE electrolyzer cells was measured and modelled. From the analysis of water transport mechanisms in SPE water electrolysis, the water-flow rate through membrane can be described by the electro-osmotic drag. The calculated electro-osmotic drag coefficients, n d, for the membrane in SPE electrolysis cells at different temperatures were compared with literature and in good agreement with those of Ge et al. and Ise et al. To describe the water-flow rate through membrane more accurately, a linear fit of n d as a function of temperature for the membrane in SPE water electrolysis was proposed in this paper. This paper studied the membrane water-flow rate experimentally and mathematically, which is of importance in the designing and optimization of the process of SPE water electrolysis. This paper also provided a novel method for measuring the electro-osmotic drag coefficient of Nafion membrane in contact with liquid water, acid and methanol solutions, etc.

  17. The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

    NASA Technical Reports Server (NTRS)

    Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  18. The Effect of Acute Microgravity on Mechanically-Induced Membrane Damage and Membrane-Membrane Fusion Events

    NASA Technical Reports Server (NTRS)

    Clarke, Mark, S. F.; Vanderburg, Charles R.; Feedback, Daniel L.

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". This response is characterized by both membrane rupture and membrane resealing events mediated by membrane-membrane fusion. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  19. Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair

    PubMed Central

    2010-01-01

    Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat. PMID:20149260

  20. H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion.

    PubMed

    Baumann, Jan; Kouassi, Nancy Mounogou; Foni, Emanuela; Klenk, Hans-Dieter; Matrosovich, Mikhail

    2016-02-01

    The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T492S, N722D, R752K, and S1132F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased

  1. H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion

    PubMed Central

    Baumann, Jan; Kouassi, Nancy Mounogou; Foni, Emanuela; Klenk, Hans-Dieter

    2015-01-01

    ABSTRACT The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions—T492S, N722D, R752K, and S1132F—were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCE Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in

  2. Self-assembled tethered bimolecular lipid membranes.

    PubMed

    Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

    2009-01-01

    This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules

  3. Effect of organic matter to nitrogen ratio on membrane bioreactor performance.

    PubMed

    Hao, L; Liao, B Q

    2015-01-01

    Effect of chemical oxygen demand (COD) to nitrogen (COD:N) ratio in feed on the performance of aerobic membrane bioreactor (MBR) for treating a synthetic high-strength industrial waste water containing glucose was studied for over 370 days. The widely recommended nutrients ratio (COD:N:P = 100:5:1) is not necessary for aerobic biological industrial waste water treatment. An increased COD:N ratio from 100:5 to 100:2.5 and 100:1.8 had a limited impact on COD removal efficiency and further led to a significant improvement in membrane performance, a reduced sludge yield, and improved effluent quality in terms of residual nutrients. An increased COD:N ratio will benefit the industrial waste water treatment using MBRs by reducing membrane fouling and sludge yield, saving chemical costs, and reducing secondary pollution by nutrients addition. Optimization of nutrients usage should be conducted for specific industrial waste water streams.

  4. Preparation and Characterization of Facilitated Transport Membranes Composed of Chitosan-Styrene and Chitosan-Acrylonitrile Copolymers Modified by Methylimidazolium Based Ionic Liquids for CO2 Separation from CH4 and N2

    PubMed Central

    Otvagina, Ksenia V.; Mochalova, Alla E.; Sazanova, Tatyana S.; Petukhov, Anton N.; Moskvichev, Alexandr A.; Vorotyntsev, Andrey V.; Afonso, Carlos A. M.; Vorotyntsev, Ilya V.

    2016-01-01

    CO2 separation was found to be facilitated by transport membranes based on novel chitosan (CS)–poly(styrene) (PS) and chitosan (CS)–poly(acrylonitrile) (PAN) copolymer matrices doped with methylimidazolium based ionic liquids: [bmim][BF4], [bmim][PF6], and [bmim][Tf2N] (IL). CS plays the role of biodegradable film former and selectivity promoter. Copolymers were prepared implementing the latest achievements in radical copolymerization with chosen monomers, which enabled the achievement of outstanding mechanical strength values for the CS-based membranes (75–104 MPa for CS-PAN and 69–75 MPa for CS-PS). Ionic liquid (IL) doping affected the surface and mechanical properties of the membranes as well as the gas separation properties. The highest CO2 permeability 400 Barrers belongs to CS-b-PS/[bmim][BF4]. The highest selectivity α (CO2/N2) = 15.5 was achieved for CS-b-PAN/[bmim][BF4]. The operational temperature of the membranes is under 220 °C. PMID:27294964

  5. Controlled permeation of cell membrane by single bubble acoustic cavitation

    PubMed Central

    Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

    2011-01-01

    pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

  6. Direct membrane binding by bacterial actin MreB.

    PubMed

    Salje, Jeanne; van den Ent, Fusinita; de Boer, Piet; Löwe, Jan

    2011-08-05

    Bacterial actin MreB is one of the key components of the bacterial cytoskeleton. It assembles into short filaments that lie just underneath the membrane and organize the cell wall synthesis machinery. Here we show that MreB from both T. maritima and E. coli binds directly to cell membranes. This function is essential for cell shape determination in E. coli and is proposed to be a general property of many, if not all, MreBs. We demonstrate that membrane binding is mediated by a membrane insertion loop in TmMreB and by an N-terminal amphipathic helix in EcMreB and show that purified TmMreB assembles into double filaments on a membrane surface that can induce curvature. This, the first example of a membrane-binding actin filament, prompts a fundamental rethink of the structure and dynamics of MreB filaments within cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Nitrate determination using anion exchange membrane and mid-infrared spectroscopy.

    PubMed

    Linker, Raphael; Shaviv, Avi

    2006-09-01

    This study investigates the combined use of an anion exchange membrane and transmittance mid-infrared spectroscopy for determining nitrate concentration in aqueous solutions and soil pastes. The method is based on immersing a small piece (2 cm(2)) of anion exchange membrane into 5 mL of solution or soil paste for 30 minutes, after which the membrane is removed, rinsed, and wiped dry. The absorbance spectrum of the charged membrane is then used to determine the amount of nitrate sorbed on the membrane. At the levels tested, the presence of carbonate or phosphate does not affect the nitrate sorption or the spectrum of the charged membrane in the vicinity of the nitrate band. Sulfate affects the spectrum of the charged membrane but does not prevent nitrate determination. For soil pastes, nitrate sorption is remarkably independent of the soil composition and is not affected by the level of soil constituents such as organic matter, clay, and calcium carbonate. Partial least squares analysis of the membrane spectra shows that there exists a strong correlation between the nitrate charge and the absorbance in the 1000-1070 cm(-1) interval, which includes the v(1) nitrate band located around 1040 cm(-1). The prediction errors range from 0.8 to 2.1 mueq, which, under the specific experimental conditions, corresponds to approximately 2 to 6 ppm N-NO(3)(-) on a solution basis or 2 to 5 mg [N]/kg [dry soil] on a dry soil basis.

  8. Effects of Phase Separation Behavior on Morphology and Performance of Polycarbonate Membranes

    PubMed Central

    Idris, Alamin; Man, Zakaria; Maulud, Abdulhalim S.; Khan, Muhammad Saad

    2017-01-01

    The phase separation behavior of bisphenol-A-polycarbonate (PC), dissolved in N-methyl-2-pyrrolidone and dichloromethane solvents in coagulant water, was studied by the cloud point method. The respective cloud point data were determined by titration against water at room temperature and the characteristic binodal curves for the ternary systems were plotted. Further, the physical properties such as viscosity, refractive index, and density of the solution were measured. The critical polymer concentrations were determined from the viscosity measurements. PC/NMP and PC/DCM membranes were fabricated by the dry-wet phase inversion technique and characterized for their morphology, structure, and thermal stability using field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis, respectively. The membranes’ performances were tested for their permeance to CO2, CH4, and N2 gases at 24 ± 0.5 °C with varying feed pressures from 2 to 10 bar. The PC/DCM membranes appeared to be asymmetric dense membrane types with appreciable thermal stability, whereas the PC/NMP membranes were observed to be asymmetric with porous structures exhibiting 4.18% and 9.17% decrease in the initial and maximum degradation temperatures, respectively. The ideal CO2/N2 and CO2/CH4 selectivities of the PC/NMP membrane decreased with the increase in feed pressures, while for the PC/DCM membrane, the average ideal CO2/N2 and CO2/CH4 selectivities were found to be 25.1 ± 0.8 and 21.1 ± 0.6, respectively. Therefore, the PC/DCM membranes with dense morphologies are appropriate for gas separation applications. PMID:28379173

  9. Conformational transition of membrane-associated terminally-acylated HIV-1 Nef

    PubMed Central

    Akgun, Bulent; Satija, Sushil; Nanda, Hirsh; Pirrone, Gregory F.; Shi, Xiaomeng; Engen, John R.; Kent, Michael S.

    2013-01-01

    Many proteins are post-translationally modified by acylation targetting them to lipid membranes. While methods such as X-ray crystallography and NMR are available to determine the structure of folded proteins in solution, the precise position of folded domains relative to a membrane remains largely unknown. We used neutron and X-ray reflection methods to measure the displacement of the core domain of HIV Nef from lipid membranes upon insertion of the N-terminal myristate group. Nef is one of several HIV-1 accessory proteins and an essential factor in AIDS progression. Upon insertion of the myristate and residues from the N-terminal arm, Nef transitions from a closed to open conformation that positions the core domain 70 Å from the lipid headgroups. This work rules out speculation that the Nef core remains closely associated with the membrane to optimize interactions with the cytoplasmic domain of MHC-1. PMID:24035710

  10. The N-Terminal Amphipathic Helix of the Topological Specificity Factor MinE Is Associated with Shaping Membrane Curvature

    PubMed Central

    Shih, Yu-Ling; Huang, Kai-Fa; Lai, Hsin-Mei; Liao, Jiahn-Haur; Lee, Chai-Siah; Chang, Chiao-Min; Mak, Huey-Ming; Hsieh, Cheng-Wei; Lin, Chu-Chi

    2011-01-01

    Pole-to-pole oscillations of the Min proteins in Escherichia coli are required for the proper placement of the division septum. Direct interaction of MinE with the cell membrane is critical for the dynamic behavior of the Min system. In vitro, this MinE-membrane interaction led to membrane deformation; however, the underlying mechanism remained unclear. Here we report that MinE-induced membrane deformation involves the formation of an amphipathic helix of MinE2–9, which, together with the adjacent basic residues, function as membrane anchors. Biochemical evidence suggested that the membrane association induces formation of the helix, with the helical face, consisting of A2, L3, and F6, inserted into the membrane. Insertion of this helix into the cell membrane can influence local membrane curvature and lead to drastic changes in membrane topology. Accordingly, MinE showed characteristic features of protein-induced membrane tubulation and lipid clustering in in vitro reconstituted systems. In conclusion, MinE shares common protein signatures with a group of membrane trafficking proteins in eukaryotic cells. These MinE signatures appear to affect membrane curvature. PMID:21738659

  11. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  12. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin.

    PubMed

    Hicks, G R; Rayle, D L; Jones, A M; Lomax, T L

    1989-07-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  13. Efficient photocatalytic degradation of gaseous N,N-dimethylformamide in tannery waste gas using doubly open-ended Ag/TiO2 nanotube array membranes

    NASA Astrophysics Data System (ADS)

    Zhao, Yang; Ma, Lin; Chang, Wenkai; Huang, Zhiding; Feng, Xugen; Qi, Xiaoxia; Li, Zenghe

    2018-06-01

    Gaseous N,N-dimethylformamide (DMF), typical volatile organic compound exhausted from manufacturing factories, may damage the health of workers under long-term exposure even at low levels. The defined geometry, porous surface and highly ordered channels make the free-standing anodic TiO2 nanotube (TiNT) arrays particularly suitable for applications of practical air purification by flow-through photocatalysis. In the present work, crystallized doubly open-ended Ag/TiNT array membranes were designed and prepared by employing a lift-off process based on an anodization-annealing-anodization-etching sequence, followed by uniform Ag nanoparticles decoration. For the photocatalytic degradation of gaseous DMF at low concentration levels close to that found in realistic pollutant air, an analytical methodology for the monitoring and determination of degradation process was developed based on the coupling of headspace sampling with gas chromatography mass spectrometry (HS-GC-MS). The doubly open-ended Ag/TiNT arrays exhibited higher removal efficiency of gaseous DMF from air compared with conventional bottom-closed Ag/TiNT arrays and pure bottomless TiNT arrays. These results indicated that the photocatalytic properties of TiNT arrays were improved with the open-bottom morphology and the Ag nanoparticles decoration. Based on the analysis with GC-MS and high performance ion chromatography (HPIC), it was found that demethylation is the main pathway of DMF degradation in photocatalytic reactions. Furthermore, decontamination of actual polluted tannery waste gas collected in leather factory proved that the photocatalysis on doubly open-ended Ag/TiNT array membrane is an efficient way and a promising application to treat air contaminated by DMF despite the complexity of various volatile organic compounds.

  14. Interaction measurement of particles bound to a lipid membrane

    NASA Astrophysics Data System (ADS)

    Sarfati, Raphael; Dufresne, Eric

    2015-03-01

    The local shape and dynamics of the plasma membrane play important roles in many cellular processes. Local membrane deformations are often mediated by the adsorption of proteins (notably from the BAR family), and their subsequent self-assembly. The emerging hypothesis is that self-assembly arises from long-range interactions of individual proteins through the membrane's deformation field. We study these interactions in a model system of micron-sized colloidal particles adsorbed onto a lipid bilayer. We use fluorescent microscopy, optical tweezers and particle tracking to measure dissipative and conservative forces as a function of the separation between the particles. We find that particles are driven together with forces of order 100 fN and remain bound in a potential well with a stiffness of order 100 fN/micron.

  15. Chitin-fibroin-hydroxyapatite membrane for guided bone regeneration: micro-computed tomography evaluation in a rat model.

    PubMed

    Baek, Young-Jae; Kim, Jung-Han; Song, Jae-Min; Yoon, Sang-Yong; Kim, Hong-Sung; Shin, Sang-Hun

    2016-12-01

    In guided bone regeneration (GBR) technique, many materials have been used for improving biological effectiveness by adding on membranes. The new membrane which was constructed with chitin-fibroin-hydroxyapatite (CNF/HAP) was compared with a collagen membrane (Bio-Gide®) by means of micro-computed tomography. Fifty-four rats were used in this study. A critical-sized (8 mm) bony defect was created in the calvaria with a trephine bur. The CNF/HAP membrane was prepared by thermally induced phase separation. In the experimental group ( n  = 18), the CNF/HAP membrane was used to cover the bony defect, and in the control group ( n  = 18), a resorbable collagen membrane (Bio-Gide®) was used. In the negative control group ( n  = 18), no membrane was used. In each group, six animals were euthanized at 2, 4, and 8 weeks after surgery. The specimens were analyzed using micro-CT. Bone volume (BV) and bone mineral density (BMD) of the new bone showed significant difference between the negative control group and membrane groups ( P  < 0.05). However, between two membranes, the difference was not significant. The CNF/HAP membrane has significant effect on the new bone formation and has the potential to be applied for guided bone regeneration.

  16. Amino-functionalized surface modification of polyacrylonitrile hollow fiber-supported polydimethylsiloxane membranes

    NASA Astrophysics Data System (ADS)

    Hu, Leiqing; Cheng, Jun; Li, Yannan; Liu, Jianzhong; Zhou, Junhu; Cen, Kefa

    2017-08-01

    This study aimed to improve surface polarity of polydimethylsiloxane (PDMS) membranes and provide surface active sites which were easy to react with other chemicals. 3-Aminopropyltriethoxysilane (APTES) containing an amino group was introduced into a PDMS membrane by crosslinking to prepare polyacrylonitrile hollow fiber-supported PDMS membranes with an amino-functionalized surface. Fourier transform infrared and X-ray photoelectron spectroscopic analyses proved the existence of APTES and its amino group in the PDMS membrane. The concentration of N atoms on the PDMS membrane surface reached ∼6% when the mass ratio of APTES/PDMS oligomer in the PDMS coating solution was increased to 4/3. The water contact angle decreased from ∼114° to ∼87.5°, indicating the improved surface polarization of the PDMS membrane. The density and swelling degree of the PDMS membrane decreased and increased, respectively, with increasing APTES content in PDMS. This phenomenon increased CO2 permeability and decreased CO2/H2 selectivity, CO2/CH4 selectivity, and CO2/N2 selectivity. When the mass ratio of APTES/PDMS oligomer was increased from 0 to 4/3, the CO2 permeation rate of the hollow fiber-supported PDMS membranes initially decreased from ∼2370 GPU to ∼860 GPU and then increased to ∼2000 GPU due to the change in coating solution viscosity.

  17. Neurotensin-metabolizing peptidases in rat fundus plasma membranes.

    PubMed

    Checler, F; Barelli, H; Kwan, C Y; Kitabgi, P; Vincent, J P

    1987-08-01

    The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.

  18. Analyzing Structural Changes of Fe-N-C Cathode Catalysts in PEM Fuel Cell by Mößbauer Spectroscopy of Complete Membrane Electrode Assemblies.

    PubMed

    Kramm, Ulrike I; Lefèvre, Michel; Bogdanoff, Peter; Schmeißer, Dieter; Dodelet, Jean-Pol

    2014-11-06

    The applicability of analyzing by Mößbauer spectroscopy the structural changes of Fe-N-C catalysts that have been tested at the cathode of membrane electrode assemblies in proton exchange membrane (PEM) fuel cells is demonstrated. The Mößbauer characterization of powders of the same catalysts was recently described in our previous publication. A possible change of the iron species upon testing in fuel cell was investigated here by Mößbauer spectroscopy, energy-dispersive X-ray cross-sectional imaging, and neutron activation analysis. Our results show that the absorption probability of γ rays by the iron nuclei in Fe-N-C is strongly affected by the presence of Nafion and water content. A detailed investigation of the effect of an oxidizing treatment (1.2 V) of the non-noble cathode in PEM fuel cell indicates that the observed activity decay is mainly attributable to carbon oxidation causing a leaching of active iron sites hosted in the carbon matrix.

  19. Corner Wrinkling at a Square Membrane Due to Symmetric Mechanical Loads

    NASA Technical Reports Server (NTRS)

    Blandino, Joseph R.; Johnston, John D.; Dharamsi, Urmil K.; Brodeur, Stephen J. (Technical Monitor)

    2001-01-01

    Thin-film membrane structures are under consideration for use in many future gossamer spacecraft systems. Examples include sunshields for large aperture telescopes, solar sails, and membrane optics. The development of capabilities for testing and analyzing pre-tensioned, thin film membrane structures is an important and challenging aspect of gossamer spacecraft technology development. This paper presents results from experimental and computational studies performed to characterize the wrinkling behavior of thin-fi[m membranes under mechanical loading. The test article is a 500 mm square membrane subjected to symmetric comer loads. Data is presented for loads ranging from 0.49 N to 4.91 N. The experimental results show that as the load increases the number of wrinkles increases, while the wrinkle amplitude decreases. The computational model uses a finite element implementation of Stein-Hedgepeth membrane wrinkling theory to predict the behavior of the membrane. Comparisons were made with experimental results for the wrinkle angle and wrinkled region. There was reasonably good agreement between the measured wrinkle angle and the predicted directions of the major principle stresses. The shape of the wrinkle region predicted by the finite element model matches that observed in the experiments; however, the size of the predicted region is smaller than that determined in the experiments.

  20. Sheet Membrane Spacesuit Water Membrane Evaporator

    NASA Technical Reports Server (NTRS)

    Bue, Grant; Trevino, Luis; Zapata, Felipe; Dillion, Paul; Castillo, Juan; Vonau, Walter; Wilkes, Robert; Vogel, Matthew; Frodge, Curtis

    2013-01-01

    A document describes a sheet membrane spacesuit water membrane evaporator (SWME), which allows for the use of one common water tank that can supply cooling water to the astronaut and to the evaporator. Test data showed that heat rejection performance dropped only 6 percent after being subjected to highly contaminated water. It also exhibited robustness with respect to freezing and Martian atmospheric simulation testing. Water was allowed to freeze in the water channels during testing that simulated a water loop failure and vapor backpressure valve failure. Upon closing the backpressure valve and energizing the pump, the ice eventually thawed and water began to flow with no apparent damage to the sheet membrane. The membrane evaporator also serves to de-gas the water loop from entrained gases, thereby eliminating the need for special degassing equipment such as is needed by the current spacesuit system. As water flows through the three annular water channels, water evaporates with the vapor flowing across the hydrophobic, porous sheet membrane to the vacuum side of the membrane. The rate at which water evaporates, and therefore, the rate at which the flowing water is cooled, is a function of the difference between the water saturation pressure on the water side of the membrane, and the pressure on the vacuum side of the membrane. The primary theory is that the hydrophobic sheet membrane retains water, but permits vapor pass-through when the vapor side pressure is less than the water saturation pressure. This results in evaporative cooling of the remaining water.

  1. Enhancement of fuel cell performance with less-water dependent composite membranes having polyoxometalate anchored nanofibrous interlayer

    NASA Astrophysics Data System (ADS)

    Abouzari-lotf, Ebrahim; Jacob, Mohan V.; Ghassemi, Hossein; Ahmad, Arshad; Nasef, Mohamed Mahmoud; Zakeri, Masoumeh; Mehdipour-Ataei, Shahram

    2016-09-01

    Polyoxometalate immobilized nanofiber was used to fabricate low gas permeable layer for composite membranes designed for proton exchange membrane fuel cell (PEMFC) operating at low relative humidity (RH). The composite membranes revealed enhanced proton conductivity in dry conditions compared with state-of-the-art pristine membrane (Nafion 112, N112). This was coupled with a low fuel crossover inheriting the composite membranes about 100 mV higher OCV than N112 when tested in PEMFC at 60 °C and 40% RH. A maximum power density of up to 930 mW cm-2 was also achieved which is substantially higher than the N112 under similar conditions (577 mW cm-2). Such remarkable performance enhancement along with undetectable leaching of immobilized polyoxometalate, high dimensional stability and low water uptake of the composite membranes suggest a strong potential for PEMFC under low RH operation.

  2. FOVEAL AVASCULAR ZONE AREA AFTER INTERNAL LIMITING MEMBRANE PEELING FOR EPIRETINAL MEMBRANE AND MACULAR HOLE COMPARED WITH THAT OF FELLOW EYES AND HEALTHY CONTROLS.

    PubMed

    Kumagai, Kazuyuki; Furukawa, Mariko; Suetsugu, Tetsuyuki; Ogino, Nobuchika

    2017-08-01

    To measure the foveal avascular zone (FAZ) area after internal limiting membrane (ILM) peeling and to determine the factors significantly correlated with the FAZ area. This was a retrospective, observational, and cross-sectional study. The affected and normal fellow eyes of 102 patients with unilateral macular diseases and 169 healthy subjects were studied. The patients underwent successful vitrectomy with internal limiting membrane peeling for an epiretinal membrane (n = 56) or a macular hole (n = 46). The superficial FAZ area and average foveal (within 1 mm) thickness were measured. The main outcome measures were the en face FAZ area measured in the optical coherence tomography angiographic images. The FAZ area in the epiretinal membrane group (0.148 ± 0.094 mm) and in the macular hole group (0.255 ± 0.111 mm) were significantly smaller than that in the healthy control group (0.358 ± 0.118 mm; all, P < 0.0001). Multiple regression analysis showed that a thicker fovea was significantly correlated with a smaller FAZ area in the epiretinal membrane group (r = -0.799, P < 0.0001), macular hole group (r = -0.473, P = 0.0042), and control group (r = -0.612, P < 0.0001). The FAZ area after internal limiting membrane peeling was smaller than that of the controls. A smaller FAZ area was correlated with a thicker fovea both in internal limiting membrane-peeled eyes and normal eyes.

  3. Fatty acids of erythrocyte membrane in acute pancreatitis patients.

    PubMed

    Kuliaviene, Irma; Gulbinas, Antanas; Cremers, Johannes; Pundzius, Juozas; Kupcinskas, Limas; Dambrauskas, Zilvinas; Jansen, Eugene

    2013-09-14

    To evaluate changes in the fatty acid composition of erythrocyte membrane phospholipids during severe and mild acute pancreatitis (AP) of alcoholic and nonalcoholic etiology. All consecutive patients with a diagnosis of AP and onset of the disease within the last 72 h admitted to the Hospital of Lithuanian University of Health Sciences between June and December 2007 were included. According to the Acute Physiology and Chronic Health Evaluation (APACHE II) scale, the patients were subdivided into the mild (APACHE II score < 7, n = 22) and severe (APACHE II score ≥ 7, n = 17) AP groups. Healthy individuals (n = 26) were enrolled as controls. Blood samples were collected from patients on admission to the hospital. Fatty acids (FAs) were extracted from erythrocyte phospholipids and expressed as percentages of the total FAs present in the chromatogram. The concentrations of superoxide dismutase and glutathione peroxidase were measured in erythrocytes. We found an increase in the percentages of saturated and monounsaturated FAs, a decrease in the percentages of total polyunsaturated FAs (PUFAs) and n-3 PUFAs in erythrocyte membrane phospholipids of AP patients compared with healthy controls. Palmitic (C16:0), palmitoleic (C16:1n7cis), arachidonic (C20:4n6), docosahexaenoic (DHA, C22:6n3), and docosapentaenoic (DPA, C22:5n3) acids were the major contributing factors. A decrease in the peroxidation and unsaturation indexes in AP patients as well as the severe and mild AP groups as compared with controls was observed. The concentrations of antioxidant enzymes in the mild AP group were lower than in the control group. In severe AP of nonalcoholic etiology, the percentages of arachidic (C20:0) and arachidonic (C20:4n6) acids were decreased as compared with the control group. The patients with mild AP of nonalcoholic etiology had the increased percentages of total saturated FAs and gama linoleic acid (C18:3n6) and the decreased percentages of elaidic (C18:1n9t

  4. Fatty acids of erythrocyte membrane in acute pancreatitis patients

    PubMed Central

    Kuliaviene, Irma; Gulbinas, Antanas; Cremers, Johannes; Pundzius, Juozas; Kupcinskas, Limas; Dambrauskas, Zilvinas; Jansen, Eugene

    2013-01-01

    AIM: To evaluate changes in the fatty acid composition of erythrocyte membrane phospholipids during severe and mild acute pancreatitis (AP) of alcoholic and nonalcoholic etiology. METHODS: All consecutive patients with a diagnosis of AP and onset of the disease within the last 72 h admitted to the Hospital of Lithuanian University of Health Sciences between June and December 2007 were included. According to the Acute Physiology and Chronic Health Evaluation (APACHE II) scale, the patients were subdivided into the mild (APACHE II score < 7, n = 22) and severe (APACHE II score ≥ 7, n = 17) AP groups. Healthy individuals (n = 26) were enrolled as controls. Blood samples were collected from patients on admission to the hospital. Fatty acids (FAs) were extracted from erythrocyte phospholipids and expressed as percentages of the total FAs present in the chromatogram. The concentrations of superoxide dismutase and glutathione peroxidase were measured in erythrocytes. RESULTS: We found an increase in the percentages of saturated and monounsaturated FAs, a decrease in the percentages of total polyunsaturated FAs (PUFAs) and n-3 PUFAs in erythrocyte membrane phospholipids of AP patients compared with healthy controls. Palmitic (C16:0), palmitoleic (C16:1n7cis), arachidonic (C20:4n6), docosahexaenoic (DHA, C22:6n3), and docosapentaenoic (DPA, C22:5n3) acids were the major contributing factors. A decrease in the peroxidation and unsaturation indexes in AP patients as well as the severe and mild AP groups as compared with controls was observed. The concentrations of antioxidant enzymes in the mild AP group were lower than in the control group. In severe AP of nonalcoholic etiology, the percentages of arachidic (C20:0) and arachidonic (C20:4n6) acids were decreased as compared with the control group. The patients with mild AP of nonalcoholic etiology had the increased percentages of total saturated FAs and gama linoleic acid (C18:3n6) and the decreased percentages of elaidic

  5. Chemical vapor deposition on chabazite (CHA) zeolite membranes for effective post-combustion CO2 capture.

    PubMed

    Kim, Eunjoo; Lee, Taehee; Kim, Hyungmin; Jung, Won-Jin; Han, Doug-Young; Baik, Hionsuck; Choi, Nakwon; Choi, Jungkyu

    2014-12-16

    Chabazite (CHA) zeolites with a pore size of 0.37 × 0.42 nm(2) are expected to separate CO2 (0.33 nm) from larger N2 (0.364 nm) in postcombustion flue gases by recognizing their minute size differences. Furthermore, the hydrophobic siliceous constituent in CHA membranes can allow for maintaining the CO2/N2 separation performance in the presence of H2O in contrast with the CO2 affinity-based membranes. In an attempt to increase the molecular sieving ability, the pore mouth size of all silica CHA (Si-CHA) particles was reduced via the chemical vapor deposition (CVD) of a silica precursor (tetraethyl orthosilicate). Accordingly, an increase of the CVD treatment duration decreased the penetration rate of CO2 into the CVD-treated Si-CHA particles. Furthermore, the CVD process was applied to siliceous CHA membranes in order to improve their CO2/N2 separation performance. Compared to the intact CHA membranes, the CO2/N2 maximum separation factor (max SF) for CVD-treated CHA membranes was increased by ∼ 2 fold under dry conditions. More desirably, the CO2/N2 max SF was increased by ∼ 3 fold under wet conditions at ∼ 50 °C, a representative temperature of the flue gas stream. In fact, the presence of H2O in the feed disfavored the permeation of N2 more than that of CO2 through CVD-modified CHA membranes and thus, contributed to the increased CO2/N2 separation factor.

  6. 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

  7. High-Tg Polynorbornene-Based Block and Random Copolymers for Butanol Pervaporation Membranes

    NASA Astrophysics Data System (ADS)

    Register, Richard A.; Kim, Dong-Gyun; Takigawa, Tamami; Kashino, Tomomasa; Burtovyy, Oleksandr; Bell, Andrew

    Vinyl addition polymers of substituted norbornene (NB) monomers possess desirably high glass transition temperatures (Tg); however, until very recently, the lack of an applicable living polymerization chemistry has precluded the synthesis of such polymers with controlled architecture, or copolymers with controlled sequence distribution. We have recently synthesized block and random copolymers of NB monomers bearing hydroxyhexafluoroisopropyl and n-butyl substituents (HFANB and BuNB) via living vinyl addition polymerization with Pd-based catalysts. Both series of polymers were cast into the selective skin layers of thin film composite (TFC) membranes, and these organophilic membranes investigated for the isolation of n-butanol from dilute aqueous solution (model fermentation broth) via pervaporation. The block copolymers show well-defined microphase-separated morphologies, both in bulk and as the selective skin layers on TFC membranes, while the random copolymers are homogeneous. Both block and random vinyl addition copolymers are effective as n-butanol pervaporation membranes, with the block copolymers showing a better flux-selectivity balance. While polyHFANB has much higher permeability and n-butanol selectivity than polyBuNB, incorporating BuNB units into the polymer (in either a block or random sequence) limits the swelling of the polyHFANB and thereby improves the n-butanol pervaporation selectivity.

  8. 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.

  9. Processing and Pretreatment Effects on Vanadium Transport in Nafion Membranes

    DOE PAGES

    Xie, Wei; Darling, Robert M.; Perry, Mike L.

    2015-10-13

    Here, this work describes how manufacturing processes and pretreatments affect the proton conductivity and vanadyl permeability of Nafion® and how these properties are altered by running in a cell. Five Nafion membranes were examined: reinforced XL100, dispersion cast NR211 and NR212, and extruded N115 and N117. The membranes were subjected to pretreatments that included annealing at 120°C and immersing in ambient temperature and boiling water and sulfuric acid. Vanadyl permeability varied by ~15X with pretreatment and ~3X with manufacturing process. Variations in ionic conductivity were comparatively modest: ~1.5X with pretreatment and ~1.2X with processing. Differences in permeability can be eliminatedmore » by annealing the extruded membranes above their glass-transition temperature or by immersing in boiling sulfuric acid. The differences induced by processing and pretreatments were largely absent from membranes removed from vanadium redox cells subjected to repeated charge/discharge cycles.« less

  10. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    PubMed

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Effect of cations (Na +, Ca 2+, Fe 3+) on the conductivity of a Nafion membrane

    NASA Astrophysics Data System (ADS)

    Hongsirikarn, Kitiya; Goodwin, James G.; Greenway, Scott; Creager, Stephen

    It is known that trace amounts of cations have a detrimental effect on the liquid-phase conductivity of perfluorosulfonated membranes at room temperature. However, the conditions used were very different from typical fuel cell conditions. Recent research has shown the impact of conductivity measurement conditions on NH 4 + contaminated membranes. In this study, the impact of nonproton-containing cations (M n+ = Na +, Ca 2+, and Fe 3+) on Nafion membrane (N-211) conductivity was investigated both in deionized (DI) water at room temperature (∼25 °C) and in the gas phase at 80 °C under conditions similar to in a PEMFC. These conductivities were compared with those of Nafion membranes contaminated with NH 4 + ions. Under the same conditions, the conductivity of a metal cationic-contaminated membrane having the same proton composition (yH+m) was similar, but slightly lower than that of an NH 4 +-contaminated membrane. The conductivity in the purely H +-form of N-211 was more than 12 times greater than the M n+-form form at 25 °C in DI water. At 80 °C, the gas-phase conductivity was 6 times and 125 times greater at 100%RH and 30%RH, respectively. The quantitative results for conductivity and activation energy of contaminated membranes under typical fuel cell conditions are reported here for the first time.

  12. 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.

  13. Chlorine-Resistant Polyamide Reverse Osmosis Membrane with Monitorable and Regenerative Sacrificial Layers.

    PubMed

    Huang, Hai; Lin, Saisai; Zhang, Lin; Hou, Li'an

    2017-03-22

    Improving chlorine stability is a high priority for aromatic polyamide (PA) reverse osmosis (RO) membranes especially in long-term desalination. In this Research Article, PA RO membranes of sustainable chlorine resistance was synthesized. Glycylglycine (Gly) was grafted onto the membrane surface as a regenerative chlorine sacrificial layer, and the zeta-potential was used to monitor the membrane performance and to conduct timely regeneration operations for chlorinated Gly. The Gly-grafted PA membrane exhibited ameliorative chlorine resistance in which the N-H moiety of glycylglycine served as sacrificial pendants against chlorine attacks. Cyclic chlorination experiments, combined with FT-IR and XPS analysis, were carried out to characterize the membrane. Results indicated that the resulting N-halamines could be fast regenerated by a simple alkaline reduction step (pH 10). A synchronous relationship between the zeta-potential and the chlorination extent of the sacrificial layer was observed. This indicated that the zeta-potential can be used as an on-site sensor to conduct a timely regeneration operation. The intrinsic mechanism of the surface sacrificial process was also studied.

  14. Hydrophilicity and antifouling property of membrane materials from cellulose acetate/polyethersulfone in DMAc.

    PubMed

    Sun, Zhonghua; Chen, Fushan

    2016-10-01

    In this study, cellulose acetate (CA) was blended with polyethersulfone (PES) to endow the ultrafiltration membrane with the improved hydrophilicity and antifouling property by using N,N-dimethylacetamide (DMAc) as the solvent. The effects of blend composition and evaporation time on the mechanical strength and pure water flux were investigated. It was found that the optimal composition of the casting solution was: 18wt% (PES), 4wt% (Polyvinylpyrrolidone K30), 3wt% (CA) and 20s (Evaporation time). The characteristics of CA-PES blend membranes were investigated through the methods of contact angle goniometer, antifouling property, compatibility, thermo gravimetric analysis and SEM. The results showed that the hydrophilicity and antifouling property of CA-PES ultrafiltration membranes were enhanced in comparison with the pure PES membranes. The CA-PES membranes exhibited semi-compatibility and good thermal stability below 270°C. This study provided a potential industrial application prospect of CA-PES membranes prepared in DMAc. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

    PubMed Central

    Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

    2014-01-01

    Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

  16. Anti-Fouling Double-Skinned Forward Osmosis Membrane with Zwitterionic Brush for Oily Wastewater Treatment.

    PubMed

    Ong, Chi Siang; Al-Anzi, Bader; Lau, Woei Jye; Goh, Pei Sean; Lai, Gwo Sung; Ismail, Ahmad Fauzi; Ong, Yue Seong

    2017-07-31

    Despite its attractive features for energy saving separation, the performance of forward osmosis (FO) has been restricted by internal concentration polarization and fast fouling propensity that occur in the membrane sublayer. These problems have significantly affected the membrane performance when treating highly contaminated oily wastewater. In this study, a novel double-skinned FO membrane with excellent anti-fouling properties has been developed for emulsified oil-water treatment. The double-skinned FO membrane comprises a fully porous sublayer sandwiched between a highly dense polyamide (PA) layer for salt rejection and a fairly loose dense bottom zwitterionic layer for emulsified oil particle removal. The top dense PA layer was synthesized via interfacial polymerization meanwhile the bottom layer was made up of a zwitterionic polyelectrolyte brush - (poly(3-(N-2-methacryloxyethyl-N,N-dimethyl) ammonatopropanesultone), abbreviated as PMAPS layer. The resultant double-skinned membrane exhibited a high water flux of 13.7 ± 0.3 L/m 2 .h and reverse salt transport of 1.6 ± 0.2 g/m 2 .h under FO mode using 2 M NaCl as the draw solution and emulsified oily solution as the feed. The double-skinned membrane outperforms the single-skinned membrane with much lower fouling propensity for emulsified oil-water separation.

  17. Gas separation mechanism of CO 2 selective amidoxime-poly(1-trimethylsilyl-1-propyne) membranes

    DOE PAGES

    Feng, Hongbo; Hong, Tao; Mahurin, Shannon Mark; ...

    2017-05-09

    Polymeric membranes for CO 2 separation have drawn significant attention in academia and industry. We prepared amidoxime-functionalized poly(1-trimethylsilyl-1-propyne) (AO-PTMSP) membranes through hydrosilylation and post-polymerization modification. Compared to neat PTMSP membranes, the AO-PTMSP membranes showed significant enhancements in CO 2/N 2 gas separation performance (CO 2 permeability ~6000 Barrer; CO 2/N 2 selectivity 17). This systematic study provides clear guidelines on how to tune the CO 2-philicity within PTMSP matrices and the effects on gas selectivity. Key parameters for elucidating the gas transport mechanism were discussed based on CO 2 sorption measurements and fractional free volume estimates. The effect of themore » AO content on CO 2/N 2 selectivity was further examined by means of density functional theory calculations. Here, both experimental and theoretical data provide consistent results that conclusively show that CO 2/N 2 separation performance is enhanced by increased CO 2 polymer interactions.« less

  18. Gas separation mechanism of CO 2 selective amidoxime-poly(1-trimethylsilyl-1-propyne) membranes

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

    Feng, Hongbo; Hong, Tao; Mahurin, Shannon Mark

    Polymeric membranes for CO 2 separation have drawn significant attention in academia and industry. We prepared amidoxime-functionalized poly(1-trimethylsilyl-1-propyne) (AO-PTMSP) membranes through hydrosilylation and post-polymerization modification. Compared to neat PTMSP membranes, the AO-PTMSP membranes showed significant enhancements in CO 2/N 2 gas separation performance (CO 2 permeability ~6000 Barrer; CO 2/N 2 selectivity 17). This systematic study provides clear guidelines on how to tune the CO 2-philicity within PTMSP matrices and the effects on gas selectivity. Key parameters for elucidating the gas transport mechanism were discussed based on CO 2 sorption measurements and fractional free volume estimates. The effect of themore » AO content on CO 2/N 2 selectivity was further examined by means of density functional theory calculations. Here, both experimental and theoretical data provide consistent results that conclusively show that CO 2/N 2 separation performance is enhanced by increased CO 2 polymer interactions.« less

  19. Hydroxyapatite-doped polycaprolactone nanofiber membrane improves tendon-bone interface healing for anterior cruciate ligament reconstruction.

    PubMed

    Han, Fei; Zhang, Peng; Sun, Yaying; Lin, Chao; Zhao, Peng; Chen, Jiwu

    2015-01-01

    Hamstring tendon autograft is a routine graft for anterior cruciate ligament (ACL) reconstruction. However, ways of improving the healing between the tendon and bone is often overlooked in clinical practice. This issue can be addressed by using a biomimetic scaffold. Herein, a biomimetic nanofiber membrane of polycaprolactone/nanohydroxyapatite/collagen (PCL/nHAp/Col) is fabricated that mimics the composition of native bone tissue for promoting tendon-bone healing. This membrane has good cytocompatibility, allowing for osteoblast cell adhesion and growth and bone formation. As a result, MC3T3 cells reveal a higher mineralization level in PCL/nHAp/Col membrane compared with PCL membrane alone. Further in vivo studies in ACL reconstruction in a rabbit model shows that PCL/nHAp/Col-wrapped tendon may afford superior tissue integration to nonwrapped tendon in the interface between the tendon and host bone as well as improved mechanical strength. This study shows that PCL/nHAp/Col nanofiber membrane wrapping of autologous tendon is effective for improving tendon healing with host bone in ACL reconstruction.

  20. Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis

    PubMed Central

    Elgass, Kirstin D.; Gabriel, Kipros; Dougan, Gordon; Lithgow, Trevor; Heinz, Eva

    2018-01-01

    Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity. PMID:29601598

  1. Biomimetic membranes and methods of making biomimetic membranes

    DOEpatents

    Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

    2016-11-08

    The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

  2. Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin, and its metabolites ceramide and ceramide phosphate†

    PubMed Central

    Subbaiah, Papasani V.; Horvath, Peter; Achar, Srinivasa B.

    2006-01-01

    Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids were similar on the phospholipase A and cholesterol esterification reactions of the enzyme, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Ceramide incorporation into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response. PMID:16605271

  3. Inorganic membranes for carbon capture and power generation

    NASA Astrophysics Data System (ADS)

    Snider, Matthew T.

    Inorganic membranes are under consideration for cost-effective reductions of carbon emissions from coal-fired power plants, both in the capture of pollutants post-firing and in the direct electrochemical conversion of coal-derived fuels for improved plant efficiency. The suitability of inorganic membrane materials for these purposes stems as much from thermal and chemical stability in coal plant operating conditions as from high performance in gas separations and power generation. Hydrophilic, micro-porous zeolite membrane structures are attractive for separating CO2 from N2 in gaseous waste streams due to the attraction of CO2 to the membrane surface and micropore walls that gives the advantage to CO2 transport. Recent studies have indicated that retention of the templating agent used in zeolite synthesis can further block N2 from the micropore interior and significantly improve CO2/N2 selectivity. However, the role of the templating agent in micro-porous transport has not been well investigated. In this work, gas sorption studies were conducted by high-pressure thermo-gravimetric analysis on Zeolite Y membrane materials to quantify the effect of the templating agent on CO2, N2, and H2O adsorption/desorption, as well as to examine the effect of humidification on overall membrane performance. In equilibrium conditions, the N2 sorption enthalpy was nearly unchanged by the presence of the templating agent, but the N2 pore occupation was reduced ˜1000x. Thus, the steric nature of the blocking of N2 from the micropores by the templating agent was confirmed. CO2 and H2O sorption enthalpies were similarly unaffected by the templating agent, and the micropore occupations were only reduced as much as the void volume taken up by the templating agent. Thus, the steric blocking effect did not occur for molecules more strongly attracted to the micropore walls. Additionally, in time-transient measurements the CO 2 and H2O mobilities were significantly enhanced by the presence

  4. Structure of the membrane domain of respiratory complex I.

    PubMed

    Efremov, Rouslan G; Sazanov, Leonid A

    2011-08-07

    Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.

  5. A novel membrane based process to isolate photosystem-I membrane complex from spinach.

    PubMed

    Liu, Jianguo; Yin, Mengmeng; Wang, Meng; Zhang, Xuefang; Ge, Baosheng; Liu, Shuang; Lu, Jianren; Cui, Zhanfeng

    2011-02-01

    The isolation of photosystem-I (PS-I) from spinach has been conducted using ultrafiltration with 300 kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on PS-I activity were optimized using parameter scanning ultrafiltration. These conditions included solution pH, ionic strength, stirring speed, and permeate flux. The effects of detergent (Triton X-100 and n-dodecyl-beta-D-maltoside) concentration on time dependent activity of PS-I were also studied using an O(2) electrode. Under optimized conditions, the PS-I purity obtained in the retentate was about 84% and the activity recovery was greater than 94% after ultrafiltration. To our knowledge, this is the first report of the isolation of a membrane protein using ultrafiltration alone.

  6. Liquid membrane purification of biogas

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

    Majumdar, S.; Guha, A.K.; Lee, Y.T.

    1991-03-01

    Conventional gas purification technologies are highly energy intensive. They are not suitable for economic removal of CO{sub 2} from methane obtained in biogas due to the small scale of gas production. Membrane separation techniques on the other hand are ideally suited for low gas production rate applications due to their modular nature. Although liquid membranes possess a high species permeability and selectivity, they have not been used for industrial applications due to the problems of membrane stability, membrane flooding and poor operational flexibility, etc. A new hollow-fiber-contained liquid membrane (HFCLM) technique has been developed recently. This technique overcomes the shortcomingsmore » of the traditional immobilized liquid membrane technology. A new technique uses two sets of hydrophobic, microporous hollow fine fibers, packed tightly in a permeator shell. The inter-fiber space is filled with an aqueous liquid acting as the membrane. The feed gas mixture is separated by selective permeation of a species through the liquid from one fiber set to the other. The second fiber set carries a sweep stream, gas or liquid, or simply the permeated gas stream. The objectives (which were met) of the present investigation were as follows. To study the selective removal of CO{sub 2} from a model biogas mixture containing 40% CO{sub 2} (the rest being N{sub 2} or CH{sub 4}) using a HFCLM permeator under various operating modes that include sweep gas, sweep liquid, vacuum and conventional permeation; to develop a mathematical model for each mode of operation; to build a large-scale purification loop and large-scale permeators for model biogas separation and to show stable performance over a period of one month.« less

  7. Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.

    PubMed

    Stoffel, Wilhelm; Jenke, Britta; Blöck, Barbara; Zumbansen, Markus; Koebke, Jürgen

    2005-03-22

    Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3(-/-) and smpd2(-/-)smpd3(-/-) double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3(-/-) mouse might mimic a form of human combined pituitary hormone deficiency.

  8. Reversible Self-Actuated Thermo-Responsive Pore Membrane

    PubMed Central

    Park, Younggeun; Gutierrez, Maria Paz; Lee, Luke P.

    2016-01-01

    Smart membranes, which can selectively control the transfer of light, air, humidity and temperature, are important to achieve indoor climate regulation. Even though reversible self-actuation of smart membranes is desirable in large-scale, reversible self-regulation remains challenging. Specifically, reversible 100% opening/closing of pore actuation showing accurate responsiveness, reproducibility and structural flexibility, including uniform structure assembly, is currently very difficult. Here, we report a reversible, thermo-responsive self-activated pore membrane that achieves opening and closing of pores. The reversible, self-actuated thermo-responsive pore membrane was fabricated with hybrid materials of poly (N-isopropylacrylamide), (PNIPAM) within polytetrafluoroethylene (PTFE) to form a multi-dimensional pore array. Using Multiphysics simulation of heat transfer and structural mechanics based on finite element analysis, we demonstrated that pore opening and closing dynamics can be self-activated at environmentally relevant temperatures. Temperature cycle characterizations of the pore structure revealed 100% opening ratio at T = 40 °C and 0% opening ratio at T = 20 °C. The flexibility of the membrane showed an accurate temperature-responsive function at a maximum bending angle of 45°. Addressing the importance of self-regulation, this reversible self-actuated thermo-responsive pore membrane will advance the development of future large-scale smart membranes needed for sustainable indoor climate control. PMID:27991563

  9. Reversible Self-Actuated Thermo-Responsive Pore Membrane

    NASA Astrophysics Data System (ADS)

    Park, Younggeun; Gutierrez, Maria Paz; Lee, Luke P.

    2016-12-01

    Smart membranes, which can selectively control the transfer of light, air, humidity and temperature, are important to achieve indoor climate regulation. Even though reversible self-actuation of smart membranes is desirable in large-scale, reversible self-regulation remains challenging. Specifically, reversible 100% opening/closing of pore actuation showing accurate responsiveness, reproducibility and structural flexibility, including uniform structure assembly, is currently very difficult. Here, we report a reversible, thermo-responsive self-activated pore membrane that achieves opening and closing of pores. The reversible, self-actuated thermo-responsive pore membrane was fabricated with hybrid materials of poly (N-isopropylacrylamide), (PNIPAM) within polytetrafluoroethylene (PTFE) to form a multi-dimensional pore array. Using Multiphysics simulation of heat transfer and structural mechanics based on finite element analysis, we demonstrated that pore opening and closing dynamics can be self-activated at environmentally relevant temperatures. Temperature cycle characterizations of the pore structure revealed 100% opening ratio at T = 40 °C and 0% opening ratio at T = 20 °C. The flexibility of the membrane showed an accurate temperature-responsive function at a maximum bending angle of 45°. Addressing the importance of self-regulation, this reversible self-actuated thermo-responsive pore membrane will advance the development of future large-scale smart membranes needed for sustainable indoor climate control.

  10. Membrane tension controls the assembly of curvature-generating proteins

    PubMed Central

    Simunovic, Mijo; Voth, Gregory A.

    2015-01-01

    Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell. PMID:26008710

  11. Piezoelectric Response of Aligned Electrospun Polyvinylidene Fluoride/Carbon Nanotube Nanofibrous Membranes.

    PubMed

    Wu, Chang-Mou; Chou, Min-Hui; Zeng, Wun-Yuan

    2018-06-10

    Polyvinylidene fluoride (PVDF) shows piezoelectricity related to its β-phase content and mechanical and electrical properties influenced by its morphology and crystallinity. Electrospinning (ES) can produce ultrafine and well-aligned PVDF nanofibers. In this study, the effects of the presence of carbon nanotubes (CNT) and optimized ES parameters on the crystal structures and piezoelectric properties of aligned PVDF/CNT nanofibrous membranes were examined. The optimal β content and piezoelectric coefficient (d 33 ) of the aligned electrospun PVDF reached 88% and 27.4 pC/N; CNT addition increased the β-phase content to 89% and d 33 to 31.3 pC/N. The output voltages of piezoelectric units with aligned electrospun PVDF/CNT membranes increased linearly with applied loading and showed good stability during cyclic dynamic compression and tension. The sensitivities of the piezoelectric units with the membranes under dynamic compression and tension were 2.26 mV/N and 4.29 mV/%, respectively. In bending tests, the output voltage increased nonlinearly with bending angle because complicated forces were involved. The output of the aligned membrane-based piezoelectric unit with CNT was 1.89 V at the bending angle of 100°. The high electric outputs indicate that the aligned electrospun PVDF/CNT membranes are potentially effective for flexible wearable sensor application with high sensitivity.

  12. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  13. Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

    PubMed

    Ho, J; Smith, S; Roh, H K

    2014-01-01

    A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.

  14. The potent effect of mycolactone on lipid membranes

    PubMed Central

    Maniti, Ofelia; Marion, Estelle; Marsollier, Laurent; Dufourc, Erick J.; Canaan, Stéphane

    2018-01-01

    Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 μM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects. PMID:29320578

  15. Thermoelectric bolometers based on silicon membranes

    NASA Astrophysics Data System (ADS)

    Varpula, Aapo; Timofeev, Andrey V.; Shchepetov, Andrey; Grigoras, Kestutis; Ahopelto, Jouni; Prunnila, Mika

    2017-05-01

    State-of-the-art high performance IR sensing and imaging systems utilize highly expensive photodetector technology, which requires exotic and toxic materials and cooling. Cost-effective alternatives, uncooled bolometer detectors, are widely used in commercial long-wave IR (LWIR) systems. Compared to the cooled detectors they are much slower and have approximately an order of magnitude lower detectivity in the LWIR. We present uncooled bolometer technology which is foreseen to be capable of narrowing the gap between the cooled and uncooled technologies. The proposed technology is based on ultra-thin silicon membranes, the thermal conductivity and electrical properties of which can be controlled by membrane thickness and doping, respectively. The thermal signal is transduced into electric voltage using thermocouple consisting of highly-doped n and p type Si beams. Reducing the thickness of the Si membrane improves the performance (i.e. sensitivity and speed) as thermal conductivity and thermal mass of Si membrane decreases with decreasing thickness. Based on experimental data we estimate the performance of these uncooled thermoelectric bolometers.

  16. Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

    PubMed

    Guo, Jinya; Miao, Yansong; Cai, Yi

    2017-01-01

    Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

  17. Size and Shape of Amyloid Fibrils Induced by Ganglioside Nanoclusters: Role of Sialyl Oligosaccharide in Fibril Formation.

    PubMed

    Matsubara, Teruhiko; Nishihara, Masaya; Yasumori, Hanaki; Nakai, Mako; Yanagisawa, Katsuhiko; Sato, Toshinori

    2017-12-05

    Ganglioside-enriched microdomains in the presynaptic neuronal membrane play a key role in the initiation of amyloid ß-protein (Aß) assembly related to Alzheimer's disease. We previously isolated lipids from a detergent-resistant membrane microdomain fraction of synaptosomes prepared from aged mouse brain and found that spherical Aß assemblies were formed on Aß-sensitive ganglioside nanoclusters (ASIGN) of reconstituted lipid bilayers in the synaptosomal fraction. In the present study, we investigated the role of oligosaccharides in Aß fibril formation induced by ganglioside-containing mixed lipid membranes that mimic the features of ASIGN. Ganglioside nanoclusters were constructed as ternary mixed lipid bilayers composed of ganglioside (GM1, GM2, GM3, GD1a, or GT1b), sphingomyelin, and cholesterol, and their surface topography was visualized by atomic force microscopy. Aß fibril formation on the nanocluster was strongly induced in the presence of 10 mol % ganglioside, and Aß-sensitive features were observed at cholesterol contents of 35-55 mol %. GM1-, GD1a-, and GT1b-containing membranes induced longer fibrils than those containing GD1b and GM2, indicating that the terminal galactose of GM1 along with N-acetylneuraminic acid accelerates protofibril elongation. These results demonstrate that Aß fibril formation is induced by ASIGN that are highly enriched ganglioside nanoclusters with a limited number of components and that the generation and elongation of Aß protofibrils are regulated by the oligosaccharide structure of gangliosides.

  18. The membrane properties and firing characteristics of rat jaw-elevator motoneurones.

    PubMed Central

    Moore, J; Appenteng, K

    1990-01-01

    1. We have determined the membrane and firing properties of fifty-six jaw-elevator motoneurones in rats that were anaesthetized with pentobarbitone, paralysed and artificially ventilated. 2. Forty-two neurones were identified as masseter motoneurones and fourteen as masseter synergist motoneurones. The membrane potentials for the sample ranged from -60 to -86 (mean = -68; S.D. = 7.3; n = 56), and spike amplitudes from 50 to 95 mV. The duration of the after-hyperpolarization following antidromic spikes in masseter motoneurones ranged from 15 to 50 ms (mean = 30; S.D. = 12.8) and their amplitudes from 1.0 to 4.5 mV (mean = 2.7; S.D. = 2.2; n = 42). 3. The mean input resistance for the total sample was 2.3 M omega (S.D. = 0.9; n = 56), membrane time constant 3.9 ms (S.D. = 0.9; n = 48) and rheobase 4.2 nA (S.D. = 2.6; n = 56). The distribution of these parameters was independent of membrane potential. We found no significant interrelationships between the membrane properties and one interpretation of this is that our sample may be drawn from a homogenous population of motoneurones. We also suggest that elevator motoneurones may have a lower Rm (specific membrane resistivity) value than cat hindlimb motoneurones because they have a similar range of input resistance values but only half the total surface area. 4. Forty-six out of forty-nine neurones fired repetitively to a depolarizing current pulse at a mean threshold of 1.6 x rheobase. Current-frequency plots were constructed for thirteen neurones and all but one showed a primary and secondary range in the firing of the first interspike interval. The mean slope in the primary range was 31 impulses s-1 nA-1 and 77 impulses s-1 nA-1 for the secondary range. The mean minimal firing frequency for steady firing was 26 impulses s-1 and, in response to an increase of stimulation, the rate increased monotonically with a slope of 11 impulses s-1 nA-1. 5. The dynamic sensitivity of twelve neurones was assessed from their

  19. Membrane order in the plasma membrane and endocytic recycling compartment.

    PubMed

    Iaea, David B; Maxfield, Frederick R

    2017-01-01

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

  20. XopN-T3SS effector of Xanthomonas axonopodis pv. punicae localizes to the plasma membrane and modulates ROS accumulation events during blight pathogenesis in pomegranate.

    PubMed

    Kumar, Rishikesh; Soni, Madhvi; Mondal, Kalyan K

    2016-12-01

    Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease of pomegranate. Xap secretes effector proteins via type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). Previously we reported that XopN, a conserved effector of Xap, modulate in planta bacterial growth, and blight disease. In continuation to that here we report the deletion of XopN from Xap caused higher accumulation of reactive oxygen species (ROS) including H 2 O 2 and O 2 - . We quantitatively assessed the higher accumulation of H 2 O 2 in pomegranate leaves infiltrated with Xap ΔxopN compared to Xap wild-type. We analysed that 1.5 to 3.3 fold increase in transcript expression of ROS and flg22-inducible genes, namely FRK1, GST1, WRKY29, PR1, PR2 and PR5 in Arabidopsis when challenged with Xap ΔxopN; contrary, the up-regulation of all the genes were compromised when challenged with either Xap wild-type or Xap ΔxopN+xopN. Further, we demonstrated the plasma-membrane based localization of XopN protein both in its natural and experimental hosts. All together, the present study suggested that XopN-T3SS effector of Xap gets localized in the plasma membrane and suppresses ROS-mediated early defense responses during blight pathogenesis in pomegranate. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. Erythrocyte Membrane Fatty Acid Composition in Premenopausal Patients with Iron Deficiency Anemia.

    PubMed

    Aktas, Mehmet; Elmastas, Mahfuz; Ozcicek, Fatih; Yilmaz, Necmettin

    2016-01-01

    Iron deficiency anemia (IDA) is one of the most common nutritional disorders in the world. In the present study, we evaluated erythrocyte membrane fatty acid composition in premenopausal patients with IDA. Blood samples of 102 premenopausal women and 88 healthy control subjects were collected. After the erythrocytes were separated from the blood samples, the membrane lipids were carefully extracted, and the various membrane fatty acids were measured by gas chromatography (GC). Statistical analyses were performed with the SPSS software program. We used blood ferritin concentration <15 ng/mL as cut-off for the diagnosis of IDA. The five most abundant individual fatty acids obtained were palmitic acid (16:0), oleic acid (18:1, n-9c), linoleic acid (18:2, n-6c), stearic acid (18:0), and erucic acid (C22:1, n-9c). These compounds constituted about 87% of the total membrane fatty acids in patients with IDA, and 79% of the total membrane fatty acids in the control group. Compared with control subjects, case patients had higher percentages of palmitic acid (29.9% case versus 25.3% control), oleic acid (16.8% case versus 15.1% control), and stearic acid (13.5% case versus 10.5% control), and lower percentages of erucic acid (11.5% case versus 13.6% control) and linoleic acid (15.2% case versus 15.4% control) in their erythrocyte membranes. In conclusion, the total-erythrocyte-membrane saturated fatty acid (SFA) composition in premenopausal women with IDA was found to be higher than that in the control group; however, the total-erythrocyte-membrane unsaturated fatty acid (UFA) composition in premenopausal women with IDA was found to be lower than that in the control group. The differences in these values were statistically significant.

  2. Nanoporous Membranes with Chemically-Tailored Pore Walls from Triblock Terpolymer Templates

    NASA Astrophysics Data System (ADS)

    Mulvenna, Ryan; Weidman, Jacob; Pople, John; Boudouris, Bryan; Phillip, William

    2014-03-01

    Membranes generated from self-assembled block polymers have shown promise as highly permeable and selective filters; however, current syntheses of such materials lack diverse pore wall chemical functionality. Here, we report the facile synthesis of polyisoprene- b-polystyrene- b-poly(N , N -dimethylacrylamide) (PI-PS-PDMA) using a controlled reversible addition-fragmentation chain transfer (RAFT) polymerization mechanism to yield a macromolecule with an easily-tunable molecular weight and a narrow molecular weight distribution. The PI-PS-PDMA is then cast into an anisotropic membrane using the self-assembly and non-solvent induced phase separation process (SNIPS) protocol. These membranes can be used in size-selective separations for particles as small as 8 nm in diameter. Furthermore, the PDMA block can be converted to poly(acrylic acid) (PAA) readily in the solid state, and this PI-PS-PAA terpolymer membrane can separate particles as low as 2 nm in diameter while still retaining a relatively high flux. This is the smallest reported separation for a block polymer-based membrane to date. Additionally, the PAA-lined pores serve as a conversion platform to be tuned to any other pore chemistry, which allows the membrane to be of great utility in optimizing chemistry-specific separations.

  3. Characterization of Polydopamine-Coated Polyethersulfone (PES) membrane for water purification

    NASA Astrophysics Data System (ADS)

    Syawaliah; Mulyati, S.; Muzaitun; Mulyasari, R.

    2018-05-01

    The polyethersulfone (PES) membrane has been prepared by phase inversion method using N-methyl-2-pyrolidone (NMP) as solvent and polydopamine (PDA) as additive. The fabricated membrane was modified by coating with PDA of 0.5 g/l concentration and 180 minutes immersion time. The characteristic of the PES membranes before and after the modification was studied in this paper. The result of the pure water permeation experiment showed that the PDA-coated PES membrane showcased a higher flux than that of pure PES membrane. Scanning Electron Microscopy (SEM) analysis confirmed that the membrane had an asymmetric structure consisting of two layers. There was no significant influence on the addition of PDA to the morphology of the pore matrix because the modification was done by surface coating. Fourier Transform Infrared Spectroscopy (FTIR) analysis showed that PDA was successfully introduced on the surface of PES membrane with the appearance of peak O-H from catechol at wavenumber of 3348 cm-1. Modification with PDA increased the mechanical strength of the membrane which affirmed by the results of the tensile and elongation at break evaluation.

  4. Membrane stabilizer

    DOEpatents

    Mingenbach, W.A.

    1988-02-09

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material. 10 figs.

  5. α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

    PubMed

    Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

    2014-06-06

    Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Membrane order in the plasma membrane and endocytic recycling compartment

    PubMed Central

    Iaea, David B.; Maxfield, Frederick R.

    2017-01-01

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

  7. Pore spanning lipid bilayers on silanised nanoporous alumina membranes

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  8. Mitochondrial lipids in Bufo arenarum full-grown oocytes.

    PubMed

    Gili, Valeria; Alonso, Telma S

    2004-05-01

    Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.

  9. Research on Hydrophilic Nature of Polyvinylpyrrolidone on Polysulfone Membrane Filtration

    NASA Astrophysics Data System (ADS)

    Tiron, L. G.; Vlad, M.; Baltă, Ş.

    2018-06-01

    The membranes used in wastewater filtration are obtained from polymers, this technique is widely applied because of the small installations and low costs as against conventional systems. The polymeric membranes have high mechanical strength and flexibility, but is a challenge to improve in the same time the permeability and retention capacity of the membranes. A process that can improve the membrane properties is the addition of additives to the polymer solution, resulting in noticeable changes in the resulting membrane structure. Polyvinylpyrrolidone (PVP) is a highly hydrophilic polymer, used as a food additive that acts as stabilizer and thickening agent, which brings improvements in membrane properties. This study analyses the effect of polyvinylpyrrolidone (PVP) on the casting solution of the prepared membranes. The polymer solution was prepared from polysulfone (PSf) and N-methyl-2-pyrrolidone (NMP) at different concentrations. The membranes were obtained by phase inversion method. The PSf/PVP/NMP membranes with different concentrations were characterized by contact angle measurements, surface roughness, morphological structure and permeation tests. The results show that the hydrophilic nature of PVP improve the pure water flux, the contact angle and exhibit a higher anti-fouling property.

  10. Dense chitosan surgical membranes produced by a coincident compression-dehydration process

    PubMed Central

    Dooley, Thomas P.; Ellis, April L.; Belousova, Maria; Petersen, Don; DeCarlo, Arthur A.

    2012-01-01

    High density chitosan membranes were produced via a novel manufacturing process for use as implantable resorbable surgical membranes. The innovative method utilizes the following three sequential steps: (1) casting an acidic chitosan solution within a silicon mold, followed by freezing; (2) neutralizing the frozen acidic chitosan solution in alkaline solution to facilitate polymerization; and (3) applying coincident compression-dehydration under a vacuum. Resulting membranes of 0.2 – 0.5 mm thickness have densities as high as 1.6 g/cm3. Inclusion of glycerol prior to the compression-dehydration step provides additional physical and clinical handling benefits. The biomaterials exhibit tensile strength with a maximum load as high as 10.9 N at ~ 2.5 mm width and clinically-relevant resistance to suture pull-out with a maximum load as high as 2.2 N. These physical properties were superior to those of a commercial reconstituted collagen membrane. The dense chitosan membranes have excellent clinical handling characteristics, such as pliability and “memory” when wet. They are semi-permeable to small molecules, biodegradable in vitro in lysozyme solution, and the rates of degradation are inversely correlated to the degree of deacetylation. Furthermore, the dense chitosan membranes are biocompatible and resorbable in vivo as demonstrated in a rat oral wound healing model. The unique combination of physical, in vitro, in vivo, and clinical handling properties demonstrate the high utility of dense chitosan membranes produced by this new method. The materials may be useful as surgical barrier membranes, scaffolds for tissue engineering, wound dressings, and as delivery devices for active ingredients. PMID:23565872

  11. Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

    PubMed

    Yavuz, Halenur; Kattan, Iman; Hernandez, Javier Matias; Hofnagel, Oliver; Witkowska, Agata; Raunser, Stefan; Walla, Peter Jomo; Jahn, Reinhard

    2018-04-17

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N-termini to their membrane-proximal C-termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates, where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Covalent attachment of phospholipid analogous polymers to modify a polymeric membrane surface: a novel approach.

    PubMed

    Xu, Zhi-Kang; Dai, Qing-Wen; Wu, Jian; Huang, Xiao-Jun; Yang, Qian

    2004-02-17

    A novel method for the surface modification of a microporous polypropylene membrane by tethering phospholipid analogous polymers (PAPs) is given, which includes the photoinduced graft polymerization of N,N-dimethylaminoethyl methacrylate (DMAEMA) and the ring-opening reaction of grafted poly-(DMAEMA) with 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes. Five 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes, containing octyloxy, dodecyloxy, tetradecyloxy, hexadecyloxy, and octadecyloxy groups in the molecular structure, were used to fabricate the PAP-modified polypropylene membranes. The attenuated total reflectance FT-IR spectra of the original, poly(DMAEMA)-grafted, and PAP-modified membranes confirmed the chemical changes on the membrane surface. Scanning electron microscope pictures showed that, compared with the original membrane, the surface porosities ofpoly(DMAEMA)-grafted and PAP-modified membranes were somewhat reduced. Water contact angles measured by the sessile drop method on PAP-modified membranes were slightly lower than that on the original polypropylene membrane, but higher than those on poly(DMAEMA)-grafted membranes with the exception of octyloxy-containing PAP-modified membranes. However, BSA adsorption experiments indicated that the five PAP-modified membranes had a much better protein-resistant property than the original polypropylene membrane and the poly(DMAEMA)-grafted membranes. For hexadecyloxy- and octadecyloxy-containing PAP-modified membranes, almost no protein adsorption was observed when the grafting degree was above 6 wt %. It was also found that the platelet adhesion was remarkably suppressed on the PAP-modified membranes. All these results demonstrate that the described approach is an effective way to improve the surface biocompatibility for polymeric membranes.

  13. Giant plasma membrane vesicles: models for understanding membrane organization.

    PubMed

    Levental, Kandice R; Levental, Ilya

    2015-01-01

    The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Synthesis and characterization of microporous inorganic membranes for propylene/propane separation

    NASA Astrophysics Data System (ADS)

    Ma, Xiaoli

    Membrane-based gas separation is promising for efficient propylene/propane (C3H6/C3H8) separation with low energy consumption and minimum environment impact. Two microporous inorganic membrane candidates, MFI-type zeolite membrane and carbon molecular sieve membrane (CMS) have demonstrated excellent thermal and chemical stability. Application of these membranes into C3H6/C3H 8 separation has not been well investigated. This dissertation presents fundamental studies on membrane synthesis, characterization and C3H 6/C3H8 separation properties of MFI zeolite membrane and CMS membrane. MFI zeolite membranes were synthesized on α-alumina supports by secondary growth method. Novel positron annihilation spectroscopy (PAS) techniques were used to non-destructively characterize the pore structure of these membranes. PAS reveals a bimodal pore structure consisting of intracrystalline zeolitic micropores of ~0.6 nm in diameter and irregular intercrystalline micropores of 1.4 to 1.8 nm in size for the membranes. The template-free synthesized membrane exhibited a high permeance but a low selectivity in C3H 6/C3H8 mixture separation. CMS membranes were synthesized by coating/pyrolysis method on mesoporous gamma-alumina support. Such supports allow coating of thin, high-quality polymer films and subsequent CMS membranes with no infiltration into support pores. The CMS membranes show strong molecular sieving effect, offering a high C3H 6/C3H8 mixture selectivity of ~30. Reduction in membrane thickness from 500 nm to 300 nm causes an increase in C3H8 permeance and He/N2 selectivity, but a decrease in the permeance of He, N 2 and C3H6 and C3H6/C 3H8 selectivity. This can be explained by the thickness dependent chain mobility of the polymer film resulting in final carbon membrane of reduced pore size with different effects on transport of gas of different sizes, including possible closure of C3H6-accessible micropores. CMS membranes demonstrate excellent C3H6/C 3H8 separation

  15. Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1.

    PubMed

    Yun, Sung Ho; Lee, Sang-Yeop; Choi, Chi-Won; Lee, Hayoung; Ro, Hyun-Joo; Jun, Sangmi; Kwon, Yong Min; Kwon, Kae Kyoung; Kim, Sang-Jin; Kim, Gun-Hwa; Kim, Seung Il

    2017-01-01

    Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV Novo ) are spherical in shape, and the average diameter of OMV Novo is 25-70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV Novo . Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

  16. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

    PubMed

    Molinos-Albert, Luis M; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Rodríguez de la Concepción, Maria Luisa; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A; Contreras, F-Xabier; Floor, Martin; Cardona, Pere J; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-13

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

  17. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

    PubMed Central

    Molinos-Albert, Luis M.; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A.; Contreras, F.-Xabier; Floor, Martin; Cardona, Pere J.; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-01

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants. PMID:28084464

  18. Method of fabricating lipid bilayer membranes on solid supports

    NASA Technical Reports Server (NTRS)

    Cho, Nam-Joon (Inventor); Frank, Curtis W. (Inventor); Glenn, Jeffrey S. (Inventor); Cheong, Kwang Ho (Inventor)

    2012-01-01

    The present invention provides a method of producing a planar lipid bilayer on a solid support. With this method, a solution of lipid vesicles is first deposited on the solid support. Next, the lipid vesicles are destabilized by adding an amphipathic peptide solution to the lipid vesicle solution. This destabilization leads to production of a planar lipid bilayer on the solid support. The present invention also provides a supported planar lipid bilayer, where the planar lipid bilayer is made of naturally occurring lipids and the solid support is made of unmodified gold or titanium oxide. Preferably, the supported planar lipid bilayer is continuous. The planar lipid bilayer may be made of any naturally occurring lipid or mixture of lipids, including, but not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinsitol, cardiolipin, cholesterol, and sphingomyelin.

  19. Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

    PubMed

    Kelly, Amélie A; Kalisch, Barbara; Hölzl, Georg; Schulze, Sandra; Thiele, Juliane; Melzer, Michael; Roston, Rebecca L; Benning, Christoph; Dörmann, Peter

    2016-09-20

    Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

  20. Micro-scale NMR Screening of New Detergents for Membrane Protein Structural Biology

    PubMed Central

    Zhang, Qinghai; Horst, Reto; Geralt, Michael; Ma, Xingquan; Hong, Wen-Xu; Finn, M. G.; Stevens, Raymond C.; Wüthrich, Kurt

    2008-01-01

    The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that micro-coil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β-barrel E.coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with μg-amounts of uniformly 2H,15N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [15N,1H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX. PMID:18479092

  1. Membrane fouling and wetting in membrane distillation and their mitigation by novel membranes with special wettability.

    PubMed

    Wang, Zhangxin; Lin, Shihong

    2017-04-01

    Membrane distillation (MD) has been identified as a promising technology to desalinate the hypersaline wastewaters from fracking and other industries. However, conventional hydrophobic MD membranes are highly susceptible to fouling and/or wetting by the hydrophobic and/or amphiphilic constituents in these wastewaters of complex compositions. This study systematically investigates the impact of the surface wetting properties on the membrane wetting and/or fouling behaviors in MD. Specifically, we compare the wetting and fouling resistance of three types of membranes of different wetting properties, including hydrophobic and omniphobic membranes as well as composite membranes with a hydrophobic substrate and a superhydrophilic top surface. We challenged the MD membranes with hypersaline feed solutions that contained a relatively high concentration of crude oil with and without added synthetic surfactants, Triton X-100. We found that the composite membranes with superhydrophilic top surface were robustly resistant to oil fouling in the absence of Triton X-100, but were subject to pore wetting in the presence of Triton X-100. On the other hand, the omniphobic membranes were easily fouled by oil-in-water emulsion without Triton X-100, but successfully sustained stable MD performance with Triton X-100 stabilized oil-in-water emulsion as the feed solution. In contrast, the conventional hydrophobic membranes failed readily regardless whether Triton X-100 was present, although via different mechanisms. These findings are corroborated by contact angle measures as well as oil-probe force spectroscopy. This study provides a holistic picture regarding how a hydrophobic membrane fails in MD and how we can leverage membranes with special wettability to prevent membrane failure in MD operations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. DDR-type zeolite membrane synthesis, modification and gas permeation studies

    DOE PAGES

    Yang, Shaowei; Cao, Zishu; Arvanitis, Antonios; ...

    2016-01-22

    DDR-type zeolite membrane was synthesized on porous α-alumina substrate by hydrothermal treatment of a ball-milled Sigmal-1 crystal seed layer in an aluminum-free precursor solution containing 1-Adamantylamine as the structure directing agent (SDA). The as-synthesized DDR zeolite membranes were defect-free but the supported zeolite layers were susceptible to crack development during the subsequent high-temperature SDA removal process. The cracks were effectively eliminated by the liquid phase chemical deposition method using tetramethoxysilane as the precursor for silica deposits. The modified membrane was extensively studied for H 2, He, O 2, N 2, CO 2, CH 4, and i-C 4H 10 pure gasmore » permeation and CO 2/CH 4 mixture separation. At 297 K and 2-bar feed gas pressure, the membrane achieved a CO 2/CH 4 separation factor of ~92 for a feed containing 90% CO 2, which decreased to 62 for a feed containing 10% CO 2 with the CO 2 permeance virtually unchanged at ~1.8×10 –7 mol/m• sup>2 s • Pa regardless of the feed composition. It also exhibited an O 2/N 2 permselectivity of 1.8 at 297 K. Furthermore, the gas permeation behaviors of the current aluminum-containing DDR type zeolite membrane are generally in good agreement with the findings in both experimental and theoretical studies on the pure-silica DDR membranes in recent literature.« less

  3. Counteracting ammonia inhibition in anaerobic digestion by removal with a hollow fiber membrane contactor.

    PubMed

    Lauterböck, B; Ortner, M; Haider, R; Fuchs, W

    2012-10-01

    The aim of the current study was to investigate the feasibility of membrane contactors for continuous ammonia (NH₃-N) removal in an anaerobic digestion process and to counteract ammonia inhibition. Two laboratory anaerobic digesters were fed slaughterhouse wastes with ammonium (NH₄⁺) concentrations ranging from 6 to 7.4 g/L. One reactor was used as reference reactor without any ammonia removal. In the second reactor, a hollow fiber membrane contactor module was used for continuous ammonia removal. The hollow fiber membranes were directly submerged into the digestate of the anaerobic reactor. Sulfuric acid was circulated in the lumen as an adsorbent solution. Using this set up, the NH₄⁺-N concentration in the membrane reactor was significantly reduced. Moreover the extraction of ammonia lowered the pH by 0.2 units. In combination that led to a lowering of the free NH₃-N concentration by about 70%. Ammonia inhibition in the reference reactor was observed when the concentration exceeded 6 g/L NH₄⁺-N or 1-1.2 g/L NH₃-N. In contrast, in the membrane reactor the volatile fatty acid concentration, an indicator for process stability, was much lower and a higher gas yield and better degradation was observed. The chosen approach offers an appealing technology to remove ammonia directly from media having high concentrations of solids and it can help to improve process efficiency in anaerobic digestion of ammonia rich substrates. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Glycosylatable GFP as a compartment-specific membrane topology reporter

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

    Lee, Hunsang; Min, Jisoo; Heijne, Gunnar von

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present amore » robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.« less

  5. Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation.

    PubMed

    Castillon, Guillaume Alain; Michon, Laetitia; Watanabe, Reika

    2013-06-01

    Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin-Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

  6. Vanadium Redox Flow Batteries Using meta-Polybenzimidazole-Based Membranes of Different Thicknesses.

    PubMed

    Noh, Chanho; Jung, Mina; Henkensmeier, Dirk; Nam, Suk Woo; Kwon, Yongchai

    2017-10-25

    15, 25, and 35 μm thick meta-polybenzimidazole (PBI) membranes are doped with H 2 SO 4 and tested in a vanadium redox flow battery (VRFB). Their performances are compared with those of Nafion membranes. Immersed in 2 M H 2 SO 4 , PBI absorbs about 2 mol of H 2 SO 4 per mole of repeat unit. This results in low conductivity and low voltage efficiency (VE). In ex-situ tests, meta-PBI shows a negligible crossover of V 3+ and V 4+ ions, much lower than that of Nafion. This is due to electrostatic repulsive forces between vanadium cations and positively charged protonated PBI backbones, and the molecular sieving effect of PBI's nanosized pores. It turns out that charge efficiency (CE) of VRFBs using meta-PBI-based membranes is unaffected by or slightly increases with decreasing membrane thickness. Thick meta-PBI membranes require about 100 mV larger potentials to achieve the same charging current as thin meta-PBI membranes. This additional potential may increase side reactions or enable more vanadium ions to overcome the electrostatic energy barrier and to enter the membrane. On this basis, H 2 SO 4 -doped meta-PBI membranes should be thin to achieve high VE and CE. The energy efficiency of 15 μm thick PBI reaches 92%, exceeding that of Nafion 212 and 117 (N212 and N117) at 40 mA cm -2 .

  7. Interactions of a hydrophobically modified polycation with zwitterionic lipid membranes.

    PubMed

    Kepczynski, Mariusz; Jamróz, Dorota; Wytrwal, Magdalena; Bednar, Jan; Rzad, Ewa; Nowakowska, Maria

    2012-01-10

    The interactions between synthetic polycations and phospholipid bilayers play an important role in some biophysical applications such as gene delivery or antibacterial usage. Despite extensive investigation into the nature of these interactions, their physical and molecular bases remain poorly understood. In this Article, we present the results of our studies on the impact of a hydrophobically modified strong polycation on the properties of a zwitterionic bilayer used as a model of the mammalian cellular membrane. The study was carried out using a set of complementary experimental methods and molecular dynamic (MD) simulations. A new polycation, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and its interactions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were examined using dynamic light scattering (DLS), zeta potential measurements, and cryo-transmission electron microscopy (cryo-TEM). Our results have shown that polymer 3 can efficiently associate with and insert into the POPC membrane. However, it does not change its lamellar structure, as was demonstrated by cryo-TEM. The influence of polymer 3 on the membrane functionality was studied by leakage experiments applying a fluorescence dye (calcein) encapsulated in the phospholipid vesicles. The MD simulations of model systems reveal that polymer 3 promotes formation of hydrophilic pores in the membrane, thus increasing considerably its permeability.

  8. Antimicrobial membrane surfaces via efficient polyethyleneimine immobilization and cationization

    NASA Astrophysics Data System (ADS)

    Qiu, Wen-Ze; Zhao, Zi-Shu; Du, Yong; Hu, Meng-Xin; Xu, Zhi-Kang

    2017-12-01

    Biofouling control is a major task in membrane separation processes for water treatment and biomedical applications. In this work, N-alkylated polyethylenimine (PEI) is facilely and efficiently introduced onto the membrane surfaces via the co-deposition of catechol (CCh) and PEI, followed by further grafting of PEIs (600 Da, 70 kDa and 750 kDa) and cationization with methyl iodide (CH3I). The physical and chemical properties of the constructed membrane surfaces are characterized with scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, zeta potential and water contact angle measurements. Antibacterial assay reveals that the optimized membrane surfaces possess around 95% antibacterial efficiency against Gram-positive Staphylococcus aureus (S. aureus) with weak adhesion of bacteria cells after 24 h of bacterial contact. Additionally, the membrane surfaces also exhibit much enhanced antifouling property during the filtration of opposite charged bovine serum albumin (BSA). These results demonstrate a useful strategy for the surface modification of separation membranes by a kind of antimicrobial and antifouling coating.

  9. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-07-29

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  10. Flow and fouling in membrane filters: Effects of membrane morphology

    NASA Astrophysics Data System (ADS)

    Sanaei, Pejman; Cummings, Linda J.

    2015-11-01

    Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

  11. A facile approach toward multifunctional polyethersulfone membranes via in situ cross-linked copolymerization.

    PubMed

    Sun, Chuangchao; Ji, Haifeng; Qin, Hui; Nie, Shengqiang; Zhao, Weifeng; Zhao, Changsheng

    2015-01-01

    In this study, multifunctional polyethersulfone (PES) membranes are prepared via in situ cross-linked copolymerization coupled with a liquid-liquid phase separation technique. Acrylic acid (AA) and N-vinylpyrrolidone (VP) are copolymerized in PES solution, and the solution is then directly used to prepare PES membranes. The infrared and X-ray photoelectron spectroscopy testing, scanning electron microscopy, and water contact angle measurements confirm the successful modification of pristine PES membrane. Protein adsorption, platelet adhesion, plasma recalcification time, and activated partial thromboplastin time assays convince that the modified PES membranes have a better biocompatibility than pristine PES membrane. In addition, the modified membranes showed good protein antifouling property and significant adsorption property of cationic dye. The loading of Ag nanoparticles into the modified membranes endows the composite membranes with antibacterial activity.

  12. The organization of LH2 complexes in membranes from Rhodobacter sphaeroides.

    PubMed

    Olsen, John D; Tucker, Jaimey D; Timney, John A; Qian, Pu; Vassilev, Cvetelin; Hunter, C Neil

    2008-11-07

    The mapping of the photosynthetic membrane of Rhodobacter sphaeroides by atomic force microscopy (AFM) revealed a unique organization of arrays of dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) core complexes surrounded and interconnected by light-harvesting LH2 complexes (Bahatyrova, S., Frese, R. N., Siebert, C. A., Olsen, J. D., van der Werf, K. O., van Grondelle, R., Niederman, R. A., Bullough, P. A., Otto, C., and Hunter, C. N. (2004) Nature 430, 1058-1062). However, membrane regions consisting solely of LH2 complexes were under-represented in these images because these small, highly curved areas of membrane rendered them difficult to image even using gentle tapping mode AFM and impossible with contact mode AFM. We report AFM imaging of membranes prepared from a mutant of R. sphaeroides, DPF2G, that synthesizes only the LH2 complexes, which assembles spherical intracytoplasmic membrane vesicles of approximately 53 nm diameter in vivo. By opening these vesicles and adsorbing them onto mica to form small, < or =120 nm, largely flat sheets we have been able to visualize the organization of these LH2-only membranes for the first time. The transition from highly curved vesicle to the planar sheet is accompanied by a change in the packing of the LH2 complexes such that approximately half of the complexes are raised off the mica surface by approximately 1 nm relative to the rest. This vertical displacement produces a very regular corrugated appearance of the planar membrane sheets. Analysis of the topographs was used to measure the distances and angles between the complexes. These data are used to model the organization of LH2 complexes in the original, curved membrane. The implications of this architecture for the light harvesting function and diffusion of quinones in native membranes of R. sphaeroides are discussed.

  13. Solid-state nanopore localization by controlled breakdown of selectively thinned membranes

    NASA Astrophysics Data System (ADS)

    Carlsen, Autumn T.; Briggs, Kyle; Hall, Adam R.; Tabard-Cossa, Vincent

    2017-02-01

    We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 × 100 nm2 precision in standard SiN x membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.

  14. Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

    PubMed

    Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

    2004-11-15

    Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

  15. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    PubMed

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  16. Transformation of metal-organic frameworks for molecular sieving membranes

    PubMed Central

    Li, Wanbin; Zhang, Yufan; Zhang, Congyang; Meng, Qin; Xu, Zehai; Su, Pengcheng; Li, Qingbiao; Shen, Chong; Fan, Zheng; Qin, Lei; Zhang, Guoliang

    2016-01-01

    The development of simple, versatile strategies for the synthesis of metal-organic framework (MOF)-derived membranes are of increasing scientific interest, but challenges exist in understanding suitable fabrication mechanisms. Here we report a route for the complete transformation of a series of MOF membranes and particles, based on multivalent cation substitution. Through our approach, the effective pore size can be reduced through the immobilization of metal salt residues in the cavities, and appropriate MOF crystal facets can be exposed, to achieve competitive molecular sieving capabilities. The method can also be used more generally for the synthesis of a variety of MOF membranes and particles. Importantly, we design and synthesize promising MOF membranes candidates that are hard to achieve through conventional methods. For example, our CuBTC/MIL-100 membrane exhibits 89, 171, 241 and 336 times higher H2 permeance than that of CO2, O2, N2 and CH4, respectively. PMID:27090597

  17. Novel ZIF-300 Mixed-Matrix Membranes for Efficient CO2 Capture.

    PubMed

    Yuan, Jianwei; Zhu, Haipeng; Sun, Jiajia; Mao, Yangyang; Liu, Gongping; Jin, Wanqin

    2017-11-08

    Because of the high separation performance and easy preparation, mixed-matrix membranes (MMMs) consisting of metal-organic frameworks have received much attention. In this article, we report a novel ZIF-300/PEBA MMM consisting of zeolite imidazolate framework (ZIF-300) crystals and polyether block amide (PEBA) matrix. The ZIF-300 crystal size was effectively reduced by optimizing the hydrothermal reaction condition from ∼15 to ∼1 μm. The morphology and physicochemical and sorption properties of the synthesized ZIF-300 crystals and as-prepared ZIF-300/PEBA MMMs were systematically studied. The results showed that ZIF-300 crystals with a size of ∼1 μm maintained excellent preferential CO 2 sorption over N 2 without degradation of the crystal structure in the MMMs. As a result, uniformly incorporated ZIF-300 crystals highly enhanced both the CO 2 permeability and the CO 2 /N 2 selectivity of pure PEBA membrane. The optimized ZIF-300-PEBA MMMs with a ZIF-300 loading of 30 wt % exhibited a high and stable CO 2 permeability of 83 Barrer and CO 2 /N 2 selectivity of 84, which are 59.2% and 53.5% higher than pure PEBA membrane, respectively. The obtained performance surpassed the upper bound of state-of-the-art membranes for CO 2 /N 2 separation. This work demonstrated that the proposed ZIF-300/PEBA MMM could be a potential candidate for an efficient CO 2 capture process.

  18. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations.

    PubMed

    Wu, Emilia L; Cheng, Xi; Jo, Sunhwan; Rui, Huan; Song, Kevin C; Dávila-Contreras, Eder M; Qi, Yifei; Lee, Jumin; Monje-Galvan, Viviana; Venable, Richard M; Klauda, Jeffery B; Im, Wonpil

    2014-10-15

    CHARMM-GUI Membrane Builder, http://www.charmm-gui.org/input/membrane, is a web-based user interface designed to interactively build all-atom protein/membrane or membrane-only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance-based algorithm for faster initial ion displacement, (5) CHARMM inputs for P21 image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM-GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. Copyright © 2014 Wiley Periodicals, Inc.

  19. Three-year experience of using venovenous extracorporeal membrane oxygenation for patients with severe respiratory failure.

    PubMed

    Ng, George W Y; Leung, Anne K H; Sin, K C; Au, S Y; Chan, Stanley C H; Chan, Osburga P K; Wu, Helen H L

    2014-10-01

    To present the 3-year experience of using venovenous extracorporeal membrane oxygenation for patients with severe respiratory failure in a single centre in Hong Kong. Case series. A 19-bed Intensive Care Unit of a tertiary hospital in Hong Kong. All patients who were managed with venovenous extracorporeal membrane oxygenation from 1 July 2010 to 30 June 2013 in the Intensive Care Unit. Overall, 31 patients (mean age, 42.2 years, standard deviation, 14.1 years; 21 males) received venovenous extracorporeal membrane oxygenation for the treatment of severe respiratory failure. Of these, 90.3% (28 patients) presented with pneumonia as the cause of the respiratory failure, and 22 of them had identifiable causes. A total of nine (29.0%) patients were diagnosed to have H1N1 infection. The median Murray score was 3.5 (interquartile range, 3.0-3.5); the median duration of venovenous extracorporeal membrane oxygenation support was 5.0 (2.8-8.6) days; and the median duration of mechanical ventilator support was 18.2 (7.8-27.9) days. The overall intensive care unit mortality was 19.4% (n=6). The overall in-hospital mortality and the 28-day mortality were both 22.6% (n=7). Among the 22 patients who had identifiable infective causes, those suffering from viral infection had lower intensive care unit and hospital mortality than those who had bacterial infection (8.3% vs 20.0%). All the H1N1 patients survived. Complications related to extracorporeal membrane oxygenation included severe bleeding (n=2; 6.5%) and mechanical complications of the circuits (n=3; 9.7%). Venovenous extracorporeal membrane oxygenation is an effective adjunctive therapy and can be used as a life-saving procedure for carefully selected patients with severe acute respiratory distress syndrome when the limits of standard therapy have been reached.

  20. Bio-reduction of N-nitrosodimethylamine (NDMA) using a hydrogen-based membrane biofilm reactor.

    PubMed

    Chung, Jinwook; Ahn, Chang-Hoon; Chen, Zhuo; Rittmann, Bruce E

    2008-01-01

    N-Nitrosodimethylamine (NDMA) is a disinfection by-product shown to be carcinogenic, mutagenic, and teratogenic. A feasible detoxification pathway for NDMA is a three-step bio-reduction that leads to ammonia and dimethylamine. This study examines the bio-reduction of NDMA in a H2-based membrane biofilm reactor (MBfR) that also is active in nitrate and sulfate reductions. In particular, the study investigates the effects of H2 availability and the relative loadings of NDMA, nitrate, and sulfate, which potentially are competing electron acceptors. The results demonstrate that NDMA was bio-reduced to a major extent (i.e., at least 96%) in a H2-based MBfR in which the electron-equivalent fluxes from H2 oxidation were dominated by nitrate and sulfate reductions. NDMA reduction kinetics responded to NDMA concentration, H2 pressure, and the presence of competing acceptors. The most important factor controlling NDMA-reduction kinetics was the H2 availability, controlled primarily by the H2 pressure, and secondarily by competition from nitrate reduction.