A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

PubMed Central

Williamson, Yulanda M.; Moura, Hercules; Simmons, Kaneatra; Whitmon, Jennifer; Melnick, Nikkol; Rees, Jon; Woolfitt, Adrian; Schieltz, David M.; Tondella, Maria L.; Ades, Edwin; Sampson, Jacquelyn; Carlone, George; Barr, John R.

2017-01-01

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria. PMID:22537821

Isolation, characterization and mode of antimicrobial action against Vibrio cholerae of methyl gallate isolated from Acacia farnesiana.

PubMed

Sánchez, E; Heredia, N; Camacho-Corona, M Del R; García, S

2013-12-01

The antimicrobial activity of Acacia farnesiana against Vibrio cholerae has been demonstrated; however, no information regarding its active compound or its mechanism of action has been documented. The active compound was isolated from A. farnesiana by bioassay-guided fractionation and identified as methyl gallate by nuclear magnetic resonance (NMR) techniques ((1) H NMR and (13) C NMR). The minimum bactericidal concentration (MBC) of methyl gallate and its effect on membrane integrity, cytoplasmic pH, membrane potential, ATP synthesis and gene expression of cholera toxin (ctx) from V. cholerae were determined. The MBC of methyl gallate ranged from 30 ± 1 to 50 ± 1 μg ml(-1) . Methyl gallate affected cell membrane integrity, causing a decrease in cytoplasmic pH (pHin , from 7·3 to <3·0), and membrane hyperpolarization, and ATP was no longer produced by the treated cells. However, methyl gallate did not affect ctx gene expression. Methyl gallate is a major antimicrobial compound from A. farnesiana that disturbs the membrane activity of V. cholerae. The effects of methyl gallate validate several traditional antimicrobial uses of A. farnesiana, and it is an attractive alternative to control V. cholerae. © 2013 The Society for Applied Microbiology.

HPLC-PFD determination of priority pollutant PAHs in water, sediment, and semipermeable membrane devices

USGS Publications Warehouse

Williamson, K.S.; Petty, J.D.; Huckins, J.N.; Lebo, J.A.; Kaiser, E.M.

2002-01-01

High performance liquid chromatography coupled with programmable fluorescence detection was employed for the determination of 15 priority pollutant polycyclic aromatic hydrocarbons (PPPAHs) in water, sediment, and semipermeable membrane devices (SPMDs). Chromatographic separation using this analytical method facilitates selectivity, sensitivity (ppt levels), and can serve as a non-destructive technique for subsequent analysis by other chromatographic and spectroscopic techniques. Extraction and sample cleanup procedures were also developed for water, sediment, and SPMDs using various chromatographic and wet chemical methods. The focus of this publication is to examine the enrichment techniques and the analytical methodologies used in the isolation, characterization, and quantitation of 15 PPPAHs in different sample matrices.

Testing of various membranes for use in a novel sediment porewater isolation chamber for infaunal invertebrate exposure to PCBs.

PubMed

Coleman, Jessica G; Lotufo, Guilherme R; Kennedy, Alan J; Poda, Aimee R; Rushing, Todd S; Ruiz, Carlos E; Bridges, Todd S

2014-07-01

In benthic sediment bioassays, determining the relative contribution to exposure by contaminants in overlying water, porewater, and sediment particles is technically challenging. The purpose of the present study was to assess the potential for membranes to be utilized as a mechanism to allow freely dissolved hydrophobic organic contaminants into a pathway isolation exposure chamber (PIC) while excluding all sediment particles and dissolved organic carbon (DOC). This investigation was conducted in support of a larger effort to assess contaminant exposure pathways to benthos. While multiple passive samplers exist for estimating concentrations of contaminants in porewater such as those using solid-phase micro extraction (SPME) and polyoxymethylene (POM), techniques to effectively isolate whole organism exposure to porewater within a sediment system are not available. We tested the use of four membranes of different pore sizes (0.1-1.2μm) including nylon, polycarbonate, polyethylsulfone, and polytetrafluoroethylene with a hydrophilic coating. Exposures included both diffusion of radiolabeled and non-labeled contaminants across membranes from aqueous, sediment slurry, and whole sediment sources to assess and evaluate the best candidate membrane. Data generated from the present study was utilized to select the most suitable membrane for use in the larger bioavailability project which sought to assess the relevance of functional ecology in bioavailability of contaminated sediments at remediation sites. The polytetrafluoroethylene membrane was selected for use in the PIC, although exclusion of dissolved organic carbon was not achieved. Published by Elsevier Ltd.

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Treesearch

Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

2001-01-01

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

Elasticity of biomembranes studied by dynamic light scattering

NASA Astrophysics Data System (ADS)

Fujime, Satoru; Miyamoto, Shigeaki

1991-05-01

Combination of osmotic swelling and dynamic light scattering makes it possible to measure the elastic modulus of biomembranes. By this technique we have observed a drastic increase in membrane flexibility on activation of Na/glucose cotransporters in membrane vesicles prepared from brush-borders of rat small intestine and on activation by micromolar [Ca2] of exocytosis in secretory granules isolated from rat pancreatic acinar cells and bovine adrenal chromaffin cells. 1 .

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers

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

Bastiaens, L.; Springael, D.; Wattiau, P.

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp.more » Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobactereium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.« less

Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers

PubMed Central

Bastiaens, Leen; Springael, Dirk; Wattiau, Pierre; Harms, Hauke; deWachter, Rupert; Verachtert, Hubert; Diels, Ludo

2000-01-01

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge. PMID:10788347

Spectroscopic characterization of cell membranes and their constituents of the plant-associated soil bacterium Azospirillum brasilense

NASA Astrophysics Data System (ADS)

Kamnev, A. A.; Antonyuk, L. P.; Matora, L. Yu.; Serebrennikova, O. B.; Sumaroka, M. V.; Colina, M.; Renou-Gonnord, M.-F.; Ignatov, V. V.

1999-05-01

Structural and compositional features of bacterial membranes and some of their isolated constituents (cell surface lipopolysaccharide, phospholipids) of the plant-growth-promoting diazotrophic rhizobacterium Azospirillum brasilense (wild-type strain Sp245) were characterized using Fourier transform infrared (FTIR) spectroscopy and some other techniques. FTIR spectra of the cell membranes were shown to comprise the main vibration modes of the relevant lipopolysaccharide and protein components which are believed to be involved in associative plant-bacterium interactions, as well as of phospholipid constituents. The role and functions of metal cations in the structural organization and physicochemical properties of bacterial cell membranes are also discussed considering their accumulation in the membranes from the culture medium.

Covalent enzyme immobilization onto carbon nanotubes using a membrane reactor

NASA Astrophysics Data System (ADS)

Voicu, Stefan Ioan; Nechifor, Aurelia Cristina; Gales, Ovidiu; Nechifor, Gheorghe

2011-05-01

Composite porous polysulfone-carbon nanotubes membranes were prepared by dispersing carbon nanotubes into a polysulfone solution followed by the membrane formation by phase inversion-immersion precipitation technique. The carbon nanotubes with amino groups on surface were functionalized with different enzymes (carbonic anhydrase, invertase, diastase) using cyanuric chloride as linker between enzyme and carbon nanotube. The composite membrane was used as a membrane reactor for a better dispersion of carbon nanotubes and access to reaction centers. The membrane also facilitates the transport of enzymes to active carbon nanotubes centers for functionalization (amino groups). The functionalized carbon nanotubes are isolated by dissolving the membranes after the end of reaction. Carbon nanotubes with covalent immobilized enzymes are used for biosensors fabrications. The obtained membranes were characterized by Scanning Electron Microscopy, Thermal analysis, FT-IR Spectroscopy, Nuclear Magnetic Resonance, and functionalized carbon nanotubes were characterized by FT-IR spectroscopy.

Multiple Gingival Recession Coverage Treated with Vestibular Incision Subperiosteal Tunnel Access Approach with or without Platelet-Rich Fibrin - A Case Series

PubMed Central

Garg, Surbhi; Arora, Sachit Anand; Chhina, Shivjot; Singh, Padam

2017-01-01

Background: Gingival recession involves both soft tissue and hard tissue loss. In this evolutionary era of dentistry, newer techniques have evolved for complete coverage of isolated recession defects. Since 2012, vestibular incision subperiosteal tunnel access (VISTA) technique was used with various regenerative membranes to treat multiple recession defects (MRDs). Platelet-rich fibrin (PRF) membrane, a pool of growth factors but have any added advantage to recession coverage techniques is controversial. Thus, in this case series, we compare the effect of VISTA with or without PRF-membrane for the treatment of Classes I and III MRDs. Subjects and Methods: Four patients between of age 30 and 40 years (two patients having bilateral Class I and another two having bilateral Class III MRDs) were selected from the Department of Periodontics, ITS Dental College, Greater Noida and designated as Case I–IV simultaneously. Recession defects at antagonist sites in each patient were corrected by VISTA approach with or without PRF-membrane. Recorded clinical parameters included recession depth, recession width, pocket probing depth, and clinical attachment level (CAL) at baseline and 6 months postoperatively. Results: Patients having Class I recession defects showed almost complete root coverage with VISTA technique alone and reflected no added advantage of PRF-membrane. However, patients with Class III recession defects treated with VISTA + PRF-membrane showed more reduction in recession depth and gain in CAL as compared to sites treated with VISTA only. Conclusion: VISTA alone is a convenient technique for treatment of Class I MRDs. Addition of PRF-membrane for Class III recession defects give better outcome in term of reduction of recession depth and gain in CAL 6 month postoperatively. PMID:29042736

Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains.

PubMed

Bong, Ji-Hong; Yoo, Gu; Park, Min; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2014-11-01

The outer membrane of Escherichia coli was previously isolated as a liposome-like outer membrane particle using an enzymatic treatment for lysozymes; for immunoassays, the particles were subsequently layered on solid supports via hydrophobic interactions. This work presents an enzyme-free isolation method for the E. coli outer membrane with autodisplayed Z-domains using ultrasonication. First, the properties of the outer membrane particle, such as the particle size, zeta potential, and total protein, were compared with the properties of particles obtained using the previous preparation methods. Compared with the conventional isolation method using an enzyme treatment, the ultrasonic method exhibited a higher efficiency at isolating the outer membrane and less contamination by cytosolic proteins. The isolated outer membrane particles were layered on a gold surface, and the roughness and thickness of the layered outer membrane layers were subsequently analyzed using AFM analysis. Finally, the antibody-binding activity of two outer membrane layers with autodisplayed Z-domains created from particles that were isolated using the enzymatic and ultrasonic isolation methods was measured using fluorescein-labeled antibody as a model analyte, and the activity of the outer membrane layer that was isolated from the ultrasonic method was estimated to be more than 20% higher than that from the conventional enzymatic method. Copyright © 2014 Elsevier Inc. All rights reserved.

Immunological properties of Micrococcus lysodeikticus membranes.

PubMed

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

1971-01-01

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

Subfractionation, characterization and in-depth proteomic analysis of glomerular membrane vesicles in human urine

PubMed Central

Hogan, Marie C.; Johnson, Kenneth L.; Zenka, Roman M.; Charlesworth, M. Cristine; Madden, Benjamin J.; Mahoney, Doug W.; Oberg, Ann L.; Huang, Bing Q.; Nesbitt, Lisa L.; Bakeberg, Jason L.; Bergen, H. Robert; Ward, Christopher J.

2014-01-01

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV) enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin positive irregularly shaped membranous vesicles and podocin/podocalyxin negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton and RhoGDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases and complement proteins involved in glomerular disease are in GMVs and some were shed in the disease state (nephrin, TRPC6 and INF2 and PLA2R). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. PMID:24196483

Engineering hybrid exosomes by membrane fusion with liposomes.

PubMed

Sato, Yuko T; Umezaki, Kaori; Sawada, Shinichi; Mukai, Sada-atsu; Sasaki, Yoshihiro; Harada, Naozumi; Shiku, Hiroshi; Akiyoshi, Kazunari

2016-02-25

Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze-thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

PubMed

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

Lipid-Based Immuno-Magnetic Separation of Archaea from a Mixed Community

NASA Astrophysics Data System (ADS)

Frickle, C. M.; Bailey, J.; Lloyd, K. G.; Shumaker, A.; Flood, B.

2014-12-01

Despite advancing techniques in microbiology, an estimated 98% of all microbial species on Earth have yet to be isolated in pure culture. Natural samples, once transferred to the lab, are commonly overgrown by "weed" species whose metabolic advantages enable them to monopolize available resources. Developing new methods for the isolation of thus-far uncultivable microorganisms would allow us to better understand their ecology, physiology and genetic potential. Physically separating target organisms from a mixed community is one approach that may allow enrichment and growth of the desired strain. Here we report on a novel method that uses known physiological variations between taxa, in this case membrane lipids, to segregate the desired organisms while keeping them alive and viable for reproduction. Magnetic antibodies bound to the molecule squalene, which is found in the cell membranes of certain archaea, but not bacteria, enable separation of archaea from bacteria in mixed samples. Viability of cells was tested by growing the separated fractions in batch culture. Efficacy and optimization of the antibody separation technique are being evaluated using qPCR and cell counts. Future work will apply this new separation technique to natural samples.

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

PubMed Central

Grabow, W O; du Preez, M

1979-01-01

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction. PMID:394678

A new method for isolation of the intraerythrocytic stages of Plasmodium and Babesia from their host cells.

PubMed

Sobolewski, B; Mackenstedt, U; Mehlhorn, H

1993-01-01

A new method for the isolation of intraerythrocytic stages of Plasmodium berghei and Babesia divergens from red blood cells is described. The technique is based on hydrodynamic forces occurring in a flow channel containing a turbulent liquid current, which are capable of rupturing infected erythrocytes and removing their plasma membrane from the parasites' surface. The temperature and the concentration of cells were revealed as factors influencing the hydrodynamic forces. About 90% of the intact and apparently infectious parasites of both species were isolated from the lysed erythrocytes.

Characterization and storage of malaria antigens: Fractionation of Plasmodium knowlesi-induced antigens of rhesus monkey erythrocyte membranes*

PubMed Central

Schmidt-Ullrich, R.; Wallach, D. F. H.; Lightholder, J.

1979-01-01

In order to characterize parasite-induced host cell membrane antigens, the plasma membranes of Plasmodium knowlesi-infected rhesus erythrocytes have been compared with those of normal red cells and purified schizonts by immunochemical and biochemical techniques. Host cell membranes and schizonts were separated by differential centrifugation following nitrogen decompression. Isolated schizonts were further fractionated into several subcellular compartments. Crossed-immune electrophoresis, against monkey anti-schizont serum, of Triton X-100-solubilized material identified 7 P. knowlesi-specific antigens, of which 4 could be detected only in the host cell membranes. These membranes also contained 3 proteins, with relative molecular masses of 55 000, 65 000 and 90 000 and isoelectric points at pH 4.5, 4.5 and 5.2, respectively, which are lacking in normal membranes. Pulse-chase experiments with (14C)-glucosamine showed that these parasite-induced host cell membrane components are glycoproteins. ImagesFig. 1Fig. 2 PMID:120762

Membrane preparation and solubilization.

PubMed

Roy, Ankita

2015-01-01

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

Synthesis of antimicrobial Nisin-phosphorylated soybean protein isolate/poly(L-lactic acid)/ZrO2 membranes.

PubMed

Jiang, Suwei; Wang, Hualin; Chu, Chenjiang; Ma, Xingkong; Sun, Min; Jiang, Shaotong

2015-01-01

Electrospinning technique was used to fabricate the model drug Nisin loaded phosphorylated soybean protein isolate/poly(l-lactic acid)/zirconium dioxide (Nisin-PSPI/PLLA/ZrO2) nanofibrous membranes. The average diameter of drug carrier PSPI/PLLA/ZrO2 nanofibers increased with the increase of content PSPI and some spindle-shape beads appeared when PSPI content reached 25 wt%. The loading dosage of Nisin caused no significant changes in the size and morphology of nanofibers when Nisin content was below 9 wt%. There existed hydrogen and Zr-O-C bonds among PSPI, PLLA and ZrO2 units, and the crystalline of PLLA matrix decreased owning to the introducing of PSPI and ZrO2 units. Moreover, the water absorption capability and degradation rate of PSPI/PLLA/ZrO2 nanofibrous membranes increased with increasing PSPI content. The antimicrobial activity and release experimental results showed that Nisin-PSPI/PLLA/ZrO2 nanofibrous membranes displayed well controlled release and better antimicrobial activity against Staphylococcus aureus (S. aureus), and the Nisin release from the medicated nanofibers could be described by Fickian diffusion model. The Nisin-PSPI/PLLA/ZrO2 nanofibrous membranes may have potential as a new nanofibrous membrane in drug delivery, food active packaging and wound dressing. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis.

PubMed Central

Zone, J J; Taylor, T B; Kadunce, D P; Meyer, L J

1990-01-01

Linear IgA bullous dermatosis (LABD) is a rare blistering skin disease characterized by basement membrane zone deposition of IgA. This study identifies a tissue antigen detected by patient serum and then isolates the autoantibody using epidermis and protein bands blotted on nitrocellulose as immunoabsorbents. Sera from 10 patients (9 with cutaneous disease and 1 with cicatrizing conjunctivitis) were evaluated. Indirect immunofluorescence revealed an IgA anti-basement membrane antibody in 6 of 10 sera with monkey esophagus substrate and 9 of 10 sera with human epidermal substrate. Immunoblotting was performed on epidermal and dermal extracts prepared from skin separated at the basement membrane zone with either sodium chloride or EDTA. Saline-separated skin expressed a 97-kD band in dermal extract alone that was recognized by 4 of 10 sera. EDTA-separated skin expressed the 97-kD band in both epidermal (4 of 10 sera) and dermal (6 of 10 sera) extract. Immunoabsorption of positive sera with epidermis purified an IgA antibody that reacted uniquely with the 97-kD band. In addition, IgA antibody bound to nitrocellulose was eluted from the 97-kD band and found to uniquely bind basement membrane zone. It is likely that the 97-kD protein identified by these techniques is responsible for basement membrane binding of IgA in LABD. Images PMID:2107211

[Isolation of circulating tumor cells in blood by means of "Isolation by SizE of Tumor cells (ISET)"].

PubMed

Liadov, V K; Skrypnikova, M A; Popova, O P

2014-01-01

There is evidence of the importance of circulating tumor cells in bloodstream as a factor of poor prognosis of cancer. The optimum method for isolating and studying of these cells is not defined. The most common methods are either based on the isolation of tumor genetic material from blood or on immune-mediated isolation of epithelial tumor cells. The first group of methods is characterized by a lack of specificity, while the latter do not allow identifying a pool of cells undergone in bloodstream epithelial-mesenchymal transformation. There is presented an overview of results of clinical trials of a new technique of isolation of tumor cells from bloodstream based on the patients' blood filtration through a membrane with defined pore sizes (ISET-Isolation by SizE of Tumor cells).

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.

PubMed

Wittig, Ilka; Karas, Michael; Schägger, Hermann

2007-07-01

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.

Integrated Disinfection Byproducts (DBPs) Mixtures Research: DBP Concentration via Reverse Osmosis Membrane Techniques

EPA Science Inventory

With the completion of the 4-lab project, the NOM concentration aspect of 4-lab is being continued with renewed focus on creating drinking water relevant freeze-dried NOM isolates that can be used for many drinking water research efforts from DBP investigations to water reuse inv...

Electrophoretic characterisation of the outer membrane proteins of Yersinia pestis isolated in north-east Brazil.

PubMed Central

Abath, F. G.; Almeida, A. M.; Ferreira, L. C.

1989-01-01

The outer membrane proteins of 38 Yersinia pestis isolates from all known plague foci of north-east Brazil were analysed by SDS-PAGE. Approximately 20 bands were consistently found in all strains analysed and 11 were selected for comparative studies. Although qualitative differences among the electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates were not observed, quantitative alterations were clearly noted for most of these proteins. No particular quantitative alteration of the electrophoretic profile of outer membrane proteins could be associated with the period of isolation and geographic origin of the isolates. The 64 kDa outer membrane protein was significantly expressed in higher amounts among Y. pestis strains isolated from a recent plague outbreak. The possible use of electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates as a tool for epidemiological studies and for the analysis of virulence determinants is discussed. Images Fig. 2 PMID:2606164

Enhanced separation of membranes during free flow zonal electrophoresis in plants.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

2007-07-15

Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

PubMed Central

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

Isolation of the Lateral Border Recycling Compartment using a diaminobenzidine-induced density shift

PubMed Central

Sullivan, David P.; Rüffer, Claas; Muller, William A.

2014-01-01

The migration of leukocytes across the endothelium and into tissue is critical to mounting an inflammatory response. The Lateral Border Recycling Compartment (LBRC), a complex vesicular-tubule invagination of the plasma membrane found at endothelial cell borders, plays an important role in the this process. Although a few proteins have been shown to be present in the LBRC, no unique marker is known. Here we detail methods that can be used to characterize a subcellular compartment that lacks an identifying marker. Initial characterization of the LBRC was performed using standard subcellular fractionation with sucrose gradients and took advantage of the observation that the compartment migrated at a lower density than other membrane compartments. To isolate larger quantities of the compartment, we modified a classic technique known as a diaminobenzidine (DAB)-induced density shift. The DAB-induced density shift allowed for specific isolation of membranes labeled with HRP conjugated antibody. Because the LBRC could be differentially labeled at 4°C and 37°C, we were able to identify proteins that are enriched in the compartment, despite lacking a unique marker. These methods serve as a model to others studying poorly characterized compartments and organelles and are applicable to a wide variety of biological systems. PMID:24915828

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

PubMed

Niemand, J; Louw, A I; Birkholtz, L; Kirk, K

2012-09-01

Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na(+) but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Isolation and Characterization of Erythrocyte and Parasite Membranes from Rhesus Red Cells Infected with P. knowlesi.

DTIC Science & Technology

1981-06-01

initiated experiments to separate and isolate the vacuolar membrane and the parasite plasma menbrane . For this, the surfaces of intact schizonts...controlled nitrogen decompression (1) is surrounded by two membranes, its own plasma membrane and the membrane of the parasitophorous vacuole. We have

Model-based diagnosis through Structural Analysis and Causal Computation for automotive Polymer Electrolyte Membrane Fuel Cell systems

NASA Astrophysics Data System (ADS)

Polverino, Pierpaolo; Frisk, Erik; Jung, Daniel; Krysander, Mattias; Pianese, Cesare

2017-07-01

The present paper proposes an advanced approach for Polymer Electrolyte Membrane Fuel Cell (PEMFC) systems fault detection and isolation through a model-based diagnostic algorithm. The considered algorithm is developed upon a lumped parameter model simulating a whole PEMFC system oriented towards automotive applications. This model is inspired by other models available in the literature, with further attention to stack thermal dynamics and water management. The developed model is analysed by means of Structural Analysis, to identify the correlations among involved physical variables, defined equations and a set of faults which may occur in the system (related to both auxiliary components malfunctions and stack degradation phenomena). Residual generators are designed by means of Causal Computation analysis and the maximum theoretical fault isolability, achievable with a minimal number of installed sensors, is investigated. The achieved results proved the capability of the algorithm to theoretically detect and isolate almost all faults with the only use of stack voltage and temperature sensors, with significant advantages from an industrial point of view. The effective fault isolability is proved through fault simulations at a specific fault magnitude with an advanced residual evaluation technique, to consider quantitative residual deviations from normal conditions and achieve univocal fault isolation.

Biosynthesis of plant cell wall polysaccharides.

PubMed

Gibeaut, D M; Carpita, N C

1994-09-01

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

A microfluidic chip integrated with a high-density PDMS-based microfiltration membrane for rapid isolation and detection of circulating tumor cells.

PubMed

Fan, Xiaoyun; Jia, Chunping; Yang, Jun; Li, Gang; Mao, Hongju; Jin, Qinghui; Zhao, Jianlong

2015-09-15

Isolation of circulating tumor cells (CTCs) by size exclusion is a widely researched technique that offers the advantage of capturing tumor cells without reliance on cell surface expression markers. In this work, we report the development of a novel polydimethylsiloxane (PDMS) membrane filter-based microdevice for rapid and highly efficient isolation of CTCs from peripheral blood. A precise and highly porous PDMS microfilter was fabricated and integrated into the microfiltration chip by combining a sacrificial transferring film with a sandwich molding method. We achieved >90% recovery when isolating lung cancer cells from spiked blood samples, with a relatively high processing throughput of 10 mL/h. In contrast to existing CTC filtration systems, which rely on low-porosity track-etch filters or expensive lithography-based filters, our microfiltration chip does not require complex e-beam lithography or the reactive ion etching process, therefore it offers a low-cost alternative tool for highly efficient CTC enrichment and in situ analysis. Thus, this new microdevice has the potential for use in routine monitoring of cancer development and cancer therapy in a clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation of Highly Purified Fractions of Plasma Membrane and Tonoplast from the Same Homogenate of Soybean Hypocotyls by Free-Flow Electrophoresis 1

PubMed Central

Sandelius, Anna Stina; Penel, Claude; Auderset, Guy; Brightman, Andrew; Millard, Merle; Morré, D. James

1986-01-01

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K+-stimulated, vanadate-inhibited Mg2+ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 9 PMID:16664771

The structure of some cytoplasmic components of plant cells in relation to the biochemical properties of isolated particles.

PubMed

HODGE, A J; MARTIN, E M; MORTON, R K

1957-01-25

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported.

THE STRUCTURE OF SOME CYTOPLASMIC COMPONENTS OF PLANT CELLS IN RELATION TO THE BIOCHEMICAL PROPERTIES OF ISOLATED PARTICLES

PubMed Central

Hodge, A. J.; Martin, E. M.; Morton, R. K.

1957-01-01

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported. PMID:13416311

Physicochemical characterization of cellulose nanocrystal and nanoporous self-assembled CNC membrane derived from Ceiba pentandra.

PubMed

Mohamed, Mohamad Azuwa; W Salleh, W N; Jaafar, Juhana; Ismail, A F; Abd Mutalib, Muhazri; Mohamad, Abu Bakar; M Zain, M F; Awang, Nor Asikin; Mohd Hir, Zul Adlan

2017-02-10

This research involves the rare utilisation of the kapok fibre (Ceiba pentandra) as a raw material for the fabrication of cellulose nanocrystal (CNC) and self-assembled CNC membranes. The isolation of CNC from Ceiba pentandra began with the extraction of cellulose via the chemical alkali extraction by using 5wt% NaOH, followed by the typical acidified bleaching method and, finally, the CNC production through acid hydrolysis with 60wt% H 2 SO 4 at the optimum time of 60min. The prepared CNC was then employed for the preparation of self-assembled membrane through the water suspension casting evaporation technique. The obtained CNC membrane was characterised in terms of its composition, crystallinity, thermal stability, as well as, structural and morphological features with the use of several techniques including FTIR, XRD, AFM, TEM, FESEM, and TGA. The FESEM and AFM analyses had illustrated the achievement of a self-assembled CNC membrane with a smooth surface and a well-distributed nano-porous structure, with the porosity of 52.82±7.79%. In addition, the findings proved that the self-assembled CNC membrane displayed good adsorption capability indicated by the recorded efficiency of 79% and 85% for 10mg/L and 5mg/L of methylene blue in an aqueous solution, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Demonstration of Erythrocyte Membrane Asymmetry.

ERIC Educational Resources Information Center

Pederson, Philip; And Others

1985-01-01

A three-period experiment was developed to help students visualize asymmetric distribution of proteins within membranes. It includes: (1) isolating erythrocyte membranes; (2) differential labeling of intact erythrocytes and isolated erythrocyte membranes with an impermeable fluorescent dye; and (3) separating proteins by polyacrylamide gel…

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis.

PubMed

Fulsundar, Shweta; Kulkarni, Heramb M; Jagannadham, Medicharla V; Nair, Rashmi; Keerthi, Sravani; Sant, Pooja; Pardesi, Karishma; Bellare, Jayesh; Chopade, Balu Ananda

2015-01-01

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neutrophilic leukocyte membrane proteins. I. Isolation.

PubMed

Hawkins, D; Sauvé, M

1978-03-01

Rabbit exudate-derived PMN were homogenized and the cell membranes isolated on a two-phase aqueous system. Glycoproteins were extracted from cell membranes with lithium diiodosalicylate. SDS polyacrylamide gel electrophoretic analysis showed a consistent pattern of three major glycoprotein entities. Cells radioiodinated supravitally showed most of the radioactivity associated with larger glycoprotein entities whereas PMN membranes radiolabeled after isolation yielded a single major peak of radioactivity associated with a much smaller protein entity. Heterologous antisera against rabbit PMN, PMN membranes, and membrane glycoproteins were all cytotoxic for PMN in the presence of complement, and all bound to the PMN surface as demonstrated with immunocolloidal gold on electron microscopy. The data suggest that one or more glycoprotein entities are membrane-associated ectoglycoproteins which can be radiolabeled supravitally.

Pore forming properties of cecropin-melittin hybrid peptide in a natural membrane.

PubMed

Milani, Alberto; Benedusi, Mascia; Aquila, Marco; Rispoli, Giorgio

2009-12-11

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH(2); CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in approximately 50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was >or=1 G Omega, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

PubMed

Goss, Reimund; Greifenhagen, Anne; Bergner, Juliane; Volke, Daniela; Hoffmann, Ralf; Wilhelm, Christian; Schaller-Laudel, Susann

2017-04-01

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Fluid control structures in microfluidic devices

DOEpatents

Mathies, Richard A.; Grover, William H.; Skelley, Alison; Lagally, Eric; Liu, Chung N.

2008-11-04

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of a variety of fluid control structures, such as structures for pumping, isolating, mixing, routing, merging, splitting, preparing, and storing volumes of fluid. The fluid control structures can be used to implement a variety of sample introduction, preparation, processing, and storage techniques.

Fluid control structures in microfluidic devices

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

Mathies, Richard A.; Grover, William H.; Skelley, Alison

2017-05-09

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of a variety of fluid control structures, such as structures for pumping, isolating, mixing, routing, merging, splitting, preparing, and storing volumes of fluid. The fluid control structures can be used to implement a variety of sample introduction, preparation, processing, and storage techniques.

Fluid control structures in microfluidic devices

NASA Technical Reports Server (NTRS)

Skelley, Alison (Inventor); Mathies, Richard A. (Inventor); Lagally, Eric (Inventor); Grover, William H. (Inventor); Liu, Chung N. (Inventor)

2008-01-01

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of a variety of fluid control structures, such as structures for pumping, isolating, mixing, routing, merging, splitting, preparing, and storing volumes of fluid. The fluid control structures can be used to implement a variety of sample introduction, preparation, processing, and storage techniques.

Visual detection technique for efficient screening and isolation of Salmonella based on a novel enrichment assay using chromatography membrane.

PubMed

Tang, F; Xiong, Y; Zhang, H; Wu, K; Xiang, Y; Shao, J-B; Ai, H-W; Xiang, Y-P; Zheng, X-L; Lv, J-R; Sun, H; Bao, L-S; Zhang, Z; Hu, H-B; Zhang, J-Y; Chen, L; Lu, J; Liu, W-Y; Mei, H; Ma, Y; Xu, C-F; Fang, A-Y; Gu, M; Xu, C-Y; Chen, Y; Chen, Z; Sun, Z-Y

2016-03-01

To detect Salmonella more efficiently and isolate strains more easily, a novel and simple detection method that uses an enrichment assay and two chromogenic reactions on a chromatography membrane was developed. Grade 3 chromatography paper is used as functionalized solid phase support (SPS), which contains specially optimized medium. One reaction for screening is based on the sulfate-reducing capacity of Salmonella. Hydrogen sulfide (H2S) generated by Salmonella reacts with ammonium ferric citrate to produce black colored ferrous sulfide. Another reaction is based on Salmonella C8 esterase that is unique for Enterobacteriaceae except Serratia and interacts with 4-methylumbelliferyl caprylate (MUCAP) to produce fluorescent umbelliferone, which is visible under ultraviolet light. A very low detection limit (10(1) CFU ml(-1)) for Salmonella was achieved on the background of 10(5) CFU ml(-1) Escherichia coli. More importantly, testing with more than 1,000 anal samples indicated that our method has a high positive detection rate and is relatively low cost, compared with the traditional culture-based method. It took only 1 day for the preliminary screening and 2 days to efficiently isolate the Salmonella cells, indicating that the new assay is specific, rapid, and simple for Salmonella detection. In contrast to the traditional culture-based method, this method can be easily used to screen and isolate targeted strains with the naked eye. The results of quantitative and comparative experiments showed that the visual detection technique is an efficient alternative method for the screening of Salmonella spp. in many applications of large-sized samples related to public health surveillance.

All-in-One Nanowire-Decorated Multifunctional Membrane for Rapid Cell Lysis and Direct DNA Isolation

PubMed Central

2015-01-01

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour. PMID:25420232

Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry.

PubMed

Le Brun, Anton P; Clifton, Luke A; Holt, Stephen A; Holden, Peter J; Lakey, Jeremy H

2016-01-01

Studying the outer membrane of Gram-negative bacteria is challenging due to the complex nature of its structure. Therefore, simplified models are required to undertake structure-function studies of processes that occur at the outer membrane/fluid interface. Model membranes can be created by immobilizing bilayers to solid supports such as gold or silicon surfaces, or as monolayers on a liquid support where the surface pressure and fluidity of the lipids can be controlled. Both model systems are amenable to having their structure probed by neutron reflectometry, a technique that provides a one-dimensional depth profile through a membrane detailing its thickness and composition. One of the strengths of neutron scattering is the ability to use contrast matching, allowing molecules containing hydrogen and those enriched with deuterium to be highlighted or matched out against the bulk isotopic composition of the solvent. Lipopolysaccharides, a major component of the outer membrane, can be isolated for incorporation into model membranes. Here, we describe the deuteration of lipopolysaccharides from rough strains of Escherichia coli for incorporation into model outer membranes, and how the use of deuterated materials enhances structural analysis of model membranes by neutron reflectometry. © 2016 Elsevier Inc. All rights reserved.

Chronic ethanol administration inhibits calmodulin-dependent Ca++ uptake in synaptosomal membranes.

PubMed

Ross, D H

1986-06-01

Chronic ethanol administration inhibits ATP-dependent Ca++ uptake in a preparation of synaptic membranes prepared from mice following 1, 4 and 7 days of ethanol exposure in a liquid diet. Addition of calmodulin (2.5 micrograms) to membranes from mice receiving the control diet produced a slight stimulation of ATP dependent Ca++ uptake. Membranes from ETOH treated mice exhibited reduced capacity to take up Ca++ in ATP-dependent fashion. When calmodulin was added to membranes isolated from mice receiving ETOH on Days 1, 4 and 7 ATP-dependent Ca++ uptake was significantly stimulated (p less than 0.01) compared to (1) ETOH treated membranes in absence of calmodulin, and (2) control membranes. Behavioral tolerance as estimated by bar holding technique was found to be 25, 65 and 91 percent complete for Days 1, 4 and 7 respectively. These studies demonstrate that continued exposure of mice to ethanol via consumption of an ethanol containing liquid diet inhibits one of the mechanisms involving the cytosolic buffering of intracellular Ca++ in nerve terminals. This biochemical effect seen in parallel with the development of tolerance to ethanol impairment of bar holding suggests that increased cytosolic Ca++ may aid in central nervous system adaptation to ethanol.

VISUALIZIATION OF CELLULAR PHOSPHOINOSITIDE POOLS WITH GFP-FUSED PROTEIN-DOMAINS

PubMed Central

Balla, Tamas; Várnai, Péter

2011-01-01

This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique. PMID:19283730

Quantitative mapping of intracellular cations in the human amniotic membrane

NASA Astrophysics Data System (ADS)

Moretto, Ph.; Llabador, Y.; Simonoff, M.; Razafindrabe, L.; Bara, M.; Guiet-Bara, A.

1993-05-01

The effect of magnesium and taurine on the permeability of cell membranes to monovalent cations has been investigated using the Bordeaux nuclear microprobe. PIXE and RBS techniques have been used to provide quantitative measurements and ion distributions in the isolated amniotic membrane. This physiological model for cellular exchanges allowed us to reveal the distribution of most elements involved in cellular pathways and the modifications under different experimental conditions of incubation in physiological fluids. The PIXE microanalysis provided an original viewpoint on these mechanisms. Following this first study, the amnion compact lamina was found to play a role which was not, up to now, taken into account in the interpretation of electrophysiological experimentations. The release of some ionic species, such as K +, from the epithelial cells, during immersion in isotonic fluids, could have been hitherto underestimated.

A novel technique to study pore-forming peptides in a natural membrane.

PubMed

Vedovato, Natascia; Rispoli, Giorgio

2007-09-01

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na(+):Ca(2+),K(+) exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K(+), Na(+) or Ca(2+)), the OS membrane resistance (R ( m )) was typically larger than 1 GOmega in the presence of 1 mM external Ca(2+). Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in approximately 50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.

Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion.

PubMed

Van Ommen Kloeke, F; Bryant, R D; Laishley, E J

1995-12-01

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

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.

Proteomic Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins*

PubMed Central

Clarke, Victoria C.; Loughlin, Patrick C.; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M.; Day, David A.; Smith, Penelope M.C.

2015-01-01

Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID:25724908

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

PubMed

Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

2009-11-15

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

Signal presequences increase mitochondrial permeability and open the multiple conductance channel.

PubMed

Kushnareva, Y E; Campo, M L; Kinnally, K W; Sokolove, P M

1999-06-01

We have reported that the signal presequence of cytochrome oxidase subunit IV from Neurospora crassa increases the permeability of isolated rat liver mitochondria [P. M. Sokolove and K. W. Kinnally (1996) Arch. Biochem. Biophys. 336, 69] and regulates the behavior of the mutiple conductance channel (MCC) of yeast inner mitochondrial membrane [T. A. Lohret and K. W. Kinnally (1995) J. Biol. Chem. 270, 15950]. Here we examine in greater detail the action of a number of mitochondrial presequences from various sources and of several control peptides on the permeability of isolated rat liver mitochondria and on MCC activity monitored via patch-clamp techniques in both mammalian mitoplasts and a reconstituted yeast system. The data indicate that the ability to alter mitochondrial permeability is a property of most, but not all, signal peptides. Furthermore, it is clear that, although signal peptides are characterized by positive charge and the ability to form amphiphilic alpha helices, these two characteristics are not sufficient to guarantee mitochondrial effects. Finally, the results reveal a strong correlation between peptide effects on the permeability of isolated mitochondria and on MCC activity: peptides that induced swelling of mouse and rat mitochondria also activated the quiescent MCC of mouse mitoplasts and induced flickering of active MCC reconstituted from yeast mitochondrial membranes. Moreover, relative peptide efficacies were very similar for mitochondrial swelling and both types of patch-clamp experiments. We propose that patch-clamp recordings of MCC activity and the high-amplitude swelling induced by signal peptides reflect the opening of a single channel. Based on the selective responsiveness of that channel to signal peptides and the dependence of its opening in isolated mitochondria on membrane potential, we further suggest that the channel is involved in the mitochondrial protein import process. Copyright 1999 Academic Press.

Topography of the Dictyostelium discoideum plasma membrane: analysis of membrane asymmetry and intermolecular disulfide bonds.

PubMed

Shiozawa, J A; Jelenska, M M; Jacobson, B S

1987-07-28

Through the application of a unique method for isolating plasma membranes, it was possible to specifically iodinate cytoplasm-exposed plasma membrane proteins in vegetative cells of the cellular slime mold Dictyostelium discoideum. The original procedure [Chaney, L. K., & Jacobson, B. S. (1983) J. Biol. Chem. 258, 10062] which involved coating cells with colloidal silica has been modified to yield a more pure preparation. The presence of the continuous and dense silica pellicle on the outside surface of the isolated plasma membrane permitted the specific labeling of cytoplasm-exposed membrane proteins. Lactoperoxidase-catalyzed iodination was employed to label cell-surface and cytoplasm-exposed membrane proteins. The isolated and radioiodinated membranes were then compared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-surface and cytoplasmic face labeling patterns were distinct. A total of 65 proteins were found to be accessible to at least one surface of the membrane. Sixteen intermolecular disulfide bond complexes were observed in the plasma membrane of Dictyostelium; most of these complexes involved glycoproteins and, hence, were exposed to the cell surface.

Liver plasma membranes: an effective method to analyze membrane proteome.

PubMed

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Protein Separation by Electrophoretic-Electroosmotic Focusing on Supported Lipid Bilayers

PubMed Central

Liu, Chunming; Monson, Christopher F.; Yang, Tinglu; Pace, Hudson; Cremer, Paul S.

2011-01-01

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch. PMID:21958061

Efficient K+ buffering by mammalian retinal glial cells is due to cooperation of specialized ion channels.

PubMed

Nilius, B; Reichenbach, A

1988-06-01

Radial glial (Müller) cells were isolated from rabbit retinae by papaine and mechanical dissociation. Regional membrane properties of these cells were studied by using the patch-clamp technique. In the course of our experiments, we found three distinct types of large K+ conducting channels. The vitread process membrane was dominated by high conductance inwardly rectifying (HCR) channels which carried, in the open state, inward currents along a conductance of about 105 pS (symmetrical solutions with 140 mM K+) but almost no outward currents. In the membrane of the soma and the proximal distal process, we found low conductance inwardly rectifying (LCR) channels which had an open state-conductance of about 60 pS and showed rather weak rectification. The endfoot membrane, on the other hand, was found to contain non-rectifying very high conductance (VHC) channels with an open state-conductance of about 360 pS (same solutions). These results suggest that mammalian Müller cells express regional membrane specializations which are optimized to carry spatial buffering currents of excess K+ ions.

Cytotopographical specialization of enzymatically isolated rabbit retinal Müller (glial) cells: K+ conductivity of the cell membrane.

PubMed

Reichenbach, A; Eberhardt, W

1988-01-01

Müller (radial glial) cells were isolated from rabbit retinae by means of papaine and mechanical dissociation. Regional membrane properties of these cells were studied by intracellular microelectrode recordings of potential responses to local application of high K+ solutions. When different parts of the cell membrane were exposed to high K+, the amplitude of the depolarizing responses varied greatly, indicating a strong regional specialization of the membrane properties. Using morphometrical data of isolated rabbit Müller cells, and a simple circuit model, we calculated the endfoot membrane to constitute more than 80% of the total K+ conductance of the cell; the specific resistivity of the endfoot membrane was about 400 omega cm2, i.e., more than 40 times less than that of the membrane of the vitread process, which is immediately adjacent. This kind of regional membrane specialization seems to be optimized in respect to the Müller cells' ability to carry spatial buffering K+ currents.

β-Cyclodextrin and permeability to water in the bladder of Bufo arenarum.

PubMed

Orce, G; Castillo, G; Chanampa, Y

2011-05-01

We measured the effect of β-cyclodextrin (BCD, a cholesterol scavenger) on water flow across the isolated toad bladder exposed to an osmotic gradient (J(w)) by a gravimetric technique. BCD, when present in the solution bathing the apical side of the bladder, inhibited the increase in J(w) caused by nystatin, a polyene antibiotic that acts by directly binding apical membrane cholesterol. When present in the basolateral bath, BCD inhibited the increase in J(w) caused by basolateral exposure to oxytocin (which binds membrane receptors and stimulates the synthesis of cAMP), but did not alter the response to theophylline (which inhibits hydrolysis of cAMP by cyclic nucleotide phosphodiesterase). The present data are consistent with the notion that agents that increase J(w) by interacting with membrane receptors, which appear to be clustered in cholesterol-rich domains of the basolateral membrane, are altered by cholesterol depletion, whereas agents that do not interact with receptors or other basolateral membrane components are not affected by this treatment. In either case, cholesterol depletion of the apical membrane does not affect the increase in J(w) brought about by an increase in intracellular cAMP concentration.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

PubMed

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

The mechanisms of renal tubule electrolyte and water absorption, 100 years after Carl Ludwig.

PubMed

Greger, R

1996-01-01

Some 154 years after Carl Ludwig's Habilitationsschrift "Contributions to the theory of the mechanism of urine secretion" renal physiology has come a long way. The mechanisms of urine formation are now understood as the result of glomerular filtration and tubule absorption of most of the filtrate. The detailed understanding of tubule transport processes has become possible with the invention of several refined techniques such as the micropuncture techniques; the microchemical analysis of nanolitre tubule fluid samples; the in vitro perfusion of isolated tubule segments of defined origin; electrophysiological analysis of electrolyte transport including micropuncture and patch-clamp techniques; transport studies in membrane vesicle preparations; recordings of intracellular electrolyte concentrations and cloning techniques of the individual membrane transport proteins. With this wealth of information we are now starting to build an integrative understanding of the function of the individual nephron segments, the regulatory processes, the integrated function of the nephron and hence the formation of the final urine. Like anatomists of previous centuries we still state that the kidney is an "organum mirable" and we recognize that basic research in this area has fertilized the analysis of the function of a large number of other organs and cells.

Systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection based on Laennec's capsule: proposal of a novel comprehensive surgical anatomy of the liver

PubMed Central

Sugioka, Atsushi; Kato, Yutaro; Tanahashi, Yoshinao

2017-01-01

Abstract Anatomical liver resection with the Glissonean pedicle isolation is widely approved as an essential procedure for safety and curability. Especially, the extrahepatic Glissonean pedicle isolation without parenchymal destruction should be an ideal procedure. However, the surgical technique has not been standardized due to a lack of anatomical understanding. Herein, we proposed a novel comprehensive surgical anatomy of the liver based on Laennec's capsule that would give a theoretical background to the extrahepatic Glissonean pedicle isolation. Laennec's capsule is the proper membrane that covers not only the entire surface of the liver including the bare area but also the intrahepatic parenchyma surrounding the Glissonean pedicles. Consequently, there exists a gap between the Glissonean pedicle and Laennec's capsule that could be reached extrahepatically and allows us to isolate the extrahepatic Glissonean pedicle without parenchymal destruction systematically. For standardization, it is essential to approach the “six gates” indicated by the “four anatomical landmarks”: the Arantius plate, the umbilical plate, the cystic plate and the Glissonean pedicle of the caudate process (G1c). This novel anatomy would contribute to standardize the surgical techniques of the systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection including laparoscopic or robotic liver resection and to bring innovative changes in hepatobiliary surgery for spreading safe and curable liver resection. PMID:28156078

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

PubMed Central

Razin, Shmuel; Hasin, Miriam; Ne'eman, Zvi; Rottem, Shlomo

1973-01-01

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with “Stubborn” disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm3. Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 μg/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily. Images PMID:4127633

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

PubMed

Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

2017-01-01

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

PubMed

Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

2017-01-01

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Microencapsulated cells as hormone delivery systems.

PubMed

Sun, A M; Goosen, M F; O'Shea, G

1987-01-01

Transplantation of pancreatic islets of Langerhans has been shown to prevent the development of many of the complications associated with diabetes. Transplanted islets, however, are readily rejected by the immune system. The use of artificial membranes to isolate the transplanted islets from the immune system of the host prolongs islet allografts in experimental animals. We have developed a method for encapsulating islets in semipermeable membranes composed of alginate and polylysine. The same technique can be applied to other endocrine cell types. The capsules are 700 to 800 micron in diameter with a hydrogel membrane approximately 4 micron thick. Intraperitoneal allografts of 5 x 10(3) encapsulated islets reversed diabetes in rats for up to 21 months and intact capsules with viable beta cells could be recovered from the recipients. Microencapsulation of endocrine cells for transplantation could potentially be used in the clinical treatment of hormone deficiency diseases.

Membrane filtration method for enumeration and isolation of Alicyclobacillus spp. from apple juice.

PubMed

Lee, S-Y; Chang, S-S; Shin, J-H; Kang, D-H

2007-11-01

To evaluate the applicability of filtration membranes for detecting Alicyclobacillus spp. spores in apple juice. Ten types of nitrocellulose membrane filters from five manufacturers were used to collect and enumerate five Alicyclobacillus spore isolates and results were compared to conventional K agar plating. Spore recovery differed among filters with an average recovery rate of 126.2%. Recovery levels also differed among spore isolates. Although significant difference (P < 0.05) in spore sizes existed, no correlation could be determined between spore size and membrane filter recovery rate. Recovery of spores using membrane filtration is dependent on the manufacturer and filter pore size. Correlations between spore recovery rate and spore size could not be determined. Low numbers of Alicyclobacillus spores in juice can be effectively detected using membrane filtration although recovery rate differences exist among different manufacturers. Use of membrane filtration is a simple, fast alternative to the week-long enrichment procedures currently employed in most quality assurance tests.

Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

PubMed Central

Haake, David A.; Matsunaga, James

2002-01-01

The outer membrane (OM) of the mammalian pathogen Leptospira kirschneri was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 μm in diameter) than the denser OMV population (0.2 to 0.3 μm in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one- and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31LipL45. A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components. PMID:12183539

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

PubMed

Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

2014-07-01

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation.

PubMed

Mathur, Chhavi; Johnson, Kory R; Tong, Brian A; Miranda, Pablo; Srikumar, Deepa; Basilio, Daniel; Latorre, Ramon; Bezanilla, Francisco; Holmgren, Miguel

2018-02-02

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker K V channel into excised squid giant axons. We found that total K + currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

[GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains].

PubMed

Markina, O V; Alekseeva, L P; Telesmanich, N R; Chemisova, O S; Akulova, M V; Markin, N V

2011-05-01

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

Reconstituting the motility of isolated intracellular cargoes.

PubMed

Hendricks, Adam G; Goldman, Yale E; Holzbaur, Erika L F

2014-01-01

Kinesin, dynein, and myosin transport intracellular cargoes including organelles, membrane-bound vesicles, and mRNA along the cytoskeleton. These motor proteins work collectively in teams to transport cargoes over long distances and navigate around obstacles in the cell. In addition, several types of motors often interact on the same cargo to allow bidirectional transport and switching between the actin and microtubule networks. To examine transport of native cargoes in a simplified in vitro system, techniques have been developed to isolate endogenous cargoes and reconstitute their motility. Isolated cargoes can be tracked and manipulated with high precision using total internal reflection fluorescence microscopy and optical trapping. Through use of native cargoes, we can examine vesicular transport in a minimal system while retaining endogenous motor stoichiometry and the biochemical and mechanical characteristics of both motor and cargo. © 2014 Elsevier Inc. All rights reserved.

A robust mass spectrometry method for rapid profiling of erythrocyte ghost membrane proteomes.

PubMed

Fye, Haddy K S; Mrosso, Paul; Bruce, Lesley; Thézénas, Marie-Laëtitia; Davis, Simon; Fischer, Roman; Rwegasira, Gration L; Makani, Julie; Kessler, Benedikt M

2018-01-01

Red blood cell (RBC) physiology is directly linked to many human disorders associated with low tissue oxygen levels or anemia including chronic obstructive pulmonary disease, congenital heart disease, sleep apnea and sickle cell anemia. Parasites such as Plasmodium spp. and phylum Apicomplexa directly target RBCs, and surface molecules within the RBC membrane are critical for pathogen interactions. Proteomics of RBC membrane 'ghost' fractions has therefore been of considerable interest, but protocols described to date are either suboptimal or too extensive to be applicable to a larger set of clinical cohorts. Here, we describe an optimised erythrocyte isolation protocol from blood, tested for various storage conditions and explored using different fractionation conditions for isolating ghost RBC membranes. Liquid chromatography mass spectrometry (LC-MS) analysis on a Q-Exactive Orbitrap instrument was used to profile proteins isolated from the comparative conditions. Data analysis was run on the MASCOT and MaxQuant platforms to assess their scope and diversity. The results obtained demonstrate a robust method for membrane enrichment enabling consistent MS based characterisation of > 900 RBC membrane proteins in single LC-MS/MS analyses. Non-detergent based membrane solubilisation methods using the tissue and supernatant fractions of isolated ghost membranes are shown to offer effective haemoglobin removal as well as diverse recovery including erythrocyte membrane proteins of high and low abundance. The methods described in this manuscript propose a medium to high throughput framework for membrane proteome profiling by LC-MS of potential applicability to larger clinical cohorts in a variety of disease contexts.

Uptake of /sup 75/Se-selenite by brush border membrane vesicles from chick duodenum stimulated by vitamin D

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

Mykkanen, H.M.; Wasserman, R.H.

1989-02-01

Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of 75Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of 75Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60-120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional ratemore » of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4-100 microM Se. 75Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 Iu, 72 h) or with 1,25(OH)2-cholecalciferol (0.5 microgram given 16 h prior to isolation of the vesicles) significantly enhanced 75Se uptake. A threefold excess of mannitol in the outside buffer reduced 75Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with 75Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with 75Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane.« less

In vitro activity of origanum vulgare essential oil against candida species

PubMed Central

Cleff, Marlete Brum; Meinerz, Ana Raquel; Xavier, Melissa; Schuch, Luiz Filipe; Schuch, Luiz Filipe; Araújo Meireles, Mário Carlos; Alves Rodrigues, Maria Regina; de Mello, João Roberto Braga

2010-01-01

The aim of this study was to evaluate the in vitro activity of the essential oil extracted from Origanum vulgare against sixteen Candida species isolates. Standard strains tested comprised C. albicans (ATCC strains 44858, 4053, 18804 and 3691), C. parapsilosis (ATCC 22019), C. krusei (ATCC 34135), C. lusitaniae (ATCC 34449) and C. dubliniensis (ATCC MY646). Six Candida albicans isolates from the vaginal mucous membrane of female dogs, one isolate from the cutaneous tegument of a dog and one isolate of a capuchin monkey were tested in parallel. A broth microdilution technique (CLSI) was used, and the inoculum concentration was adjusted to 5 x 106 CFU mL-1. The essential oil was obtained by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography. Susceptibility was expressed as Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC). All isolates tested in vitro were sensitive to O. vulgare essential oil. The chromatographic analysis revealed that the main compounds present in the essential oil were 4-terpineol (47.95%), carvacrol (9.42%), thymol (8.42%) and □-terpineol (7.57%). C. albicans isolates obtained from animal mucous membranes exhibited MIC and MFC values of 2.72 μL mL-1 and 5 μL mL-1, respectively. MIC and MFC values for C. albicans standard strains were 2.97 μL mL-1 and 3.54 μL mL-1, respectively. The MIC and MFC for non-albicans species were 2.10 μL mL-1 and 2.97 μL mL-1, respectively. The antifungal activity of O. vulgare essential oil against Candida spp. observed in vitro suggests its administration may represent an alternative treatment for candidiasis. PMID:24031471

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

PubMed

Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

2015-01-01

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Evidence for a temperature acclimation mechanism in bacteria: an empirical test of a membrane-mediated trade-off

USGS Publications Warehouse

Hall, Edward K.; Singer, Gabriel A.; Kainz, Martin J.; Lennon, Jay T.

2010-01-01

1. Shifts in bacterial community composition along temporal and spatial temperature gradients occur in a wide range of habitats and have potentially important implications for ecosystem functioning. However, it is often challenging to empirically link an adaptation or acclimation that defines environmental niche or biogeography with a quantifiable phenotype, especially in micro-organisms. 2. Here we evaluate a possible mechanistic explanation for shifts in bacterioplankton community composition in response to temperature by testing a previously hypothesized membrane mediated trade-off between resource acquisition and respiratory costs. 3. We isolated two strains of Flavobacterium sp. at two temperatures (cold isolate and warm isolate) from the epilimnion of a small temperate lake in North Central Minnesota. 4. Compared with the cold isolate the warm isolate had higher growth rate, higher carrying capacity, lower lag time and lower respiration at the high temperature and lower phosphorus uptake at the low temperature. We also observed significant differences in membrane lipid composition between isolates and between environments that were consistent with adjustments necessary to maintain membrane fluidity at different temperatures. 5. Our results suggest that temperature acclimation in planktonic bacteria is, in part, a resource-dependent membrane-facilitated phenomenon. This study provides an explicit example of how a quantifiable phenotype can be linked through physiology to competitive ability and environmental niche.

Systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection based on Laennec's capsule: proposal of a novel comprehensive surgical anatomy of the liver.

PubMed

Sugioka, Atsushi; Kato, Yutaro; Tanahashi, Yoshinao

2017-01-01

Anatomical liver resection with the Glissonean pedicle isolation is widely approved as an essential procedure for safety and curability. Especially, the extrahepatic Glissonean pedicle isolation without parenchymal destruction should be an ideal procedure. However, the surgical technique has not been standardized due to a lack of anatomical understanding. Herein, we proposed a novel comprehensive surgical anatomy of the liver based on Laennec's capsule that would give a theoretical background to the extrahepatic Glissonean pedicle isolation. Laennec's capsule is the proper membrane that covers not only the entire surface of the liver including the bare area but also the intrahepatic parenchyma surrounding the Glissonean pedicles. Consequently, there exists a gap between the Glissonean pedicle and Laennec's capsule that could be reached extrahepatically and allows us to isolate the extrahepatic Glissonean pedicle without parenchymal destruction systematically. For standardization, it is essential to approach the "six gates" indicated by the "four anatomical landmarks": the Arantius plate, the umbilical plate, the cystic plate and the Glissonean pedicle of the caudate process (G1c). This novel anatomy would contribute to standardize the surgical techniques of the systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection including laparoscopic or robotic liver resection and to bring innovative changes in hepatobiliary surgery for spreading safe and curable liver resection. © 2017 The Authors. Journal of Hepato-Biliary-Pancreatic Sciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Endothelial cell membrane vesicles in the study of organ preference of metastasis.

PubMed

Johnson, R C; Augustin-Voss, H G; Zhu, D Z; Pauli, B U

1991-01-01

Many malignancies exhibit distinct patterns of metastasis that appear to be mediated by receptor/ligand-like interactions between tumor cells and organ-specific vascular endothelium. In order to study endothelial cell surface molecules involved in the binding of metastatic cells, we developed a perfusion method to isolate outside-out membrane vesicles from the lumenal surface of rat lung microvascular endothelium. Lungs were perfused in situ for 4 h at 37 degrees C with a solution of 100 mM formaldehyde, 2 mM dithiothreitol in phosphate-buffered saline to induce endothelial cell vesiculation. Radioiodinated rat lung endothelial cell membrane vesicles bound lung-metastatic tumor cells (B16F10, R323OAC-MET) in significantly higher numbers than their low or nonmetastatic counterparts (B16F0, R323OAC-LR). In contrast, leg endothelial membrane vesicle showed no binding preference for either cell line. Neuraminidase treatment of vesicles abolished specificity of adhesion of lung-derived vesicles to lung metastatic tumor cells. These results demonstrate that in situ perfusion is an appropriate technique to obtain pure endothelial cell membrane vesicles containing functionally active adhesion molecules. The preferential binding of lung-derived endothelial cell membrane vesicles by lung metastatic tumor cells is evidence of the importance of endothelial cell adhesion molecules in the formation of metastases.

Comparative proteomic investigation of metastatic and non-metastatic osteosarcoma cells of human and canine origin

PubMed Central

Roy, Jahnabi; Wycislo, Kathryn L.; Pondenis, Holly; Fan, Timothy M.

2017-01-01

Osteosarcoma is the most common bone cancer in dogs and people. In order to improve clinical outcomes, it is necessary to identify proteins that are differentially expressed by metastatic cells. Membrane bound proteins are responsible for multiple pro-metastatic functions. Therefore characterizing the differential expression of membranous proteins between metastatic and non-metastatic clonal variants will allow the discovery of druggable targets and consequently improve treatment methodology. The objective of this investigation was to systemically identify the membrane-associated proteomics of metastatic and non-metastatic variants of human and canine origin. Two clonal variants of divergent in vivo metastatic potential from human and canine origins were used. The plasma membranes were isolated and peptide fingerprinting was used to identify differentially expressed proteins. Selected proteins were further validated using western blotting, flow cytometry, confocal microscopy and immunohistochemistry. Over 500 proteins were identified for each cell line with nearly 40% of the proteins differentially regulated. Conserved between both species, metastatic variants demonstrated significant differences in expression of membrane proteins that are responsible for pro-metastatic functions. Additionally, CD147, CD44 and vimentin were validated using various biochemical techniques. Taken together, through a comparative proteomic approach we have identified several differentially expressed cell membrane proteins that will help in the development of future therapeutics. PMID:28910304

Comparative proteomic investigation of metastatic and non-metastatic osteosarcoma cells of human and canine origin.

PubMed

Roy, Jahnabi; Wycislo, Kathryn L; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2017-01-01

Osteosarcoma is the most common bone cancer in dogs and people. In order to improve clinical outcomes, it is necessary to identify proteins that are differentially expressed by metastatic cells. Membrane bound proteins are responsible for multiple pro-metastatic functions. Therefore characterizing the differential expression of membranous proteins between metastatic and non-metastatic clonal variants will allow the discovery of druggable targets and consequently improve treatment methodology. The objective of this investigation was to systemically identify the membrane-associated proteomics of metastatic and non-metastatic variants of human and canine origin. Two clonal variants of divergent in vivo metastatic potential from human and canine origins were used. The plasma membranes were isolated and peptide fingerprinting was used to identify differentially expressed proteins. Selected proteins were further validated using western blotting, flow cytometry, confocal microscopy and immunohistochemistry. Over 500 proteins were identified for each cell line with nearly 40% of the proteins differentially regulated. Conserved between both species, metastatic variants demonstrated significant differences in expression of membrane proteins that are responsible for pro-metastatic functions. Additionally, CD147, CD44 and vimentin were validated using various biochemical techniques. Taken together, through a comparative proteomic approach we have identified several differentially expressed cell membrane proteins that will help in the development of future therapeutics.

Chapter 6: cubic membranes the missing dimension of cell membrane organization.

PubMed

Almsherqi, Zakaria A; Landh, Tomas; Kohlwein, Sepp D; Deng, Yuru

2009-01-01

Biological membranes are among the most fascinating assemblies of biomolecules: a bilayer less than 10 nm thick, composed of rather small lipid molecules that are held together simply by noncovalent forces, defines the cell and discriminates between "inside" and "outside", survival, and death. Intracellular compartmentalization-governed by biomembranes as well-is a characteristic feature of eukaryotic cells, which allows them to fulfill multiple and highly specialized anabolic and catabolic functions in strictly controlled environments. Although cellular membranes are generally visualized as flat sheets or closely folded isolated objects, multiple observations also demonstrate that membranes may fold into "unusual", highly organized structures with 2D or 3D periodicity. The obvious correlation of highly convoluted membrane organizations with pathological cellular states, for example, as a consequence of viral infection, deserves close consideration. However, knowledge about formation and function of these highly organized 3D periodic membrane structures is scarce, primarily due to the lack of appropriate techniques for their analysis in vivo. Currently, the only direct way to characterize cellular membrane architecture is by transmission electron microscopy (TEM). However, deciphering the spatial architecture solely based on two-dimensionally projected TEM images is a challenging task and prone to artifacts. In this review, we will provide an update on the current progress in identifying and analyzing 3D membrane architectures in biological systems, with a special focus on membranes with cubic symmetry, and their potential role in physiological and pathophysiological conditions. Proteomics and lipidomics approaches in defined experimental cell systems may prove instrumental to understand formation and function of 3D membrane morphologies.

Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system.

PubMed

Cowan, Lauren S; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.

The properties of single cones isolated from the tiger salamander retina

PubMed Central

Attwell, David; Werblin, Frank S.; Wilson, Martin

1982-01-01

1. The properties of isolated single cones were studied using the voltage-clamp technique, with two micro-electrodes inserted under visual control. 2. Single cones had input resistances, when impaled with two electrodes, of up to 270 MΩ. This is probably lower than the true membrane resistance, because of damage by the impaling electrodes. The cone capacitance was about 85 pF. 3. The cone membrane contains a time-dependent current, IB, controlled by voltage, and a separate photosensitive current. 4. The gated current, IB, is an inward current with a reversal potential around -25 mV. It is activated by hyperpolarization over the range -30 to -80 mV, and at constant voltage obeys first order (exponential) kinetics. The gating time constant is typically 50 ms at the resting potential of -45 mV, rises to 170 ms at -70 mV, and decreases for further hyperpolarization. 5. The spectral sensitivity curve of the cone light response peaks at 620 nm wave-length, and is narrower than the nomogram for vitamin A2-based pigments. The light responses of isolated cones are spectrally univariant. 6. Voltage-clamped photocurrents were recorded at various membrane potentials, for light steps of various intensities. The photocurrent reversed at around -8 mV. The time course of the photocurrent, for a given intensity, was approximately independent of voltage (although its magnitude was voltage-dependent). The shape of the peak current—voltage relation of the light-sensitive current was independent of light intensity (although its magnitude was intensity-dependent). 7. These results can be explained if: (a) light simply changes the number of photosensitive channels open, without altering the properties of an open channel; (b) the reactions controlling the production of internal transmitter, the binding of internal transmitter to the photosensitive channels, and the closing and opening of the channels are unaffected by the electric field in the cone membrane, even though at least some of these reactions take place in the membrane. 8. IB plays only a small role in shaping the cone voltage response to light. ImagesPlate 1 PMID:7131315

Rapid negative inotropic effect induced by TNF-α in rat heart perfused related to PKC activation.

PubMed

Jude, B; Vetel, S; Giroux-Metges, M A; Pennec, J P

2018-07-01

Myocardial depression, frequently observed in septic shock, is mediated by circulating molecules such as cytokines. TNF-α appears to be the most important pro-inflammatory cytokine released during the early phase of a septic shock. It was previously shown that TNF-α had a negative inotropic effect on myocardium. Now, the aim of this study was to investigate the effects of the activation of PKC by TNF-α on heart function, and to determine if this cytokine could induce a decrease of membrane excitability. Isolated rat hearts (n = 6) were perfused with Tyrode solution containing TNF-α at 20 ng/ml during 30 min by using a Langendorff technique. Expressions of PKC-α and PKC-ε were analysed by western blot on membrane and cytosol proteins extracted from ventricular myocardium. Patch clamp was performed on freshly isolated cardiomyocytes (n = 8). Compared to control situation, 30 min of TNF-α perfusion led to cardiac dysfunction with a decrease of the heart rate (-83%), the force (-20%) and speed of relaxation (-18%) and the coronary flow (-25%). This is associated with an activation and a membrane targeting of both PKC-α and PKC-ε isoforms in ventricle with respectively +123% and +54% compared to control hearts. Nevertheless, TNF-α had no significant effect on voltage-gated sodium current (109.0%+/- 12.5) after addition of the cytokine when compared to control. These results showed that TNF-α had a negative inotropic effect on the isolated rat heart and can induce PKC activation leading to an impaired contractility of the heart. However the early heart dysfunction induced by the cytokine was not associated to a decrease of cardiomyocytes membrane excitability as it has been evidenced in skeletal muscle fibres. Copyright © 2017 Elsevier Ltd. All rights reserved.

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture

PubMed Central

O’Connell, Ryan P.; Musa, Hassan; Gomez, Mario San Martin; Avula, Uma Mahesh; Herron, Todd J.; Kalifa, Jerome; Anumonwo, Justus M. B.

2015-01-01

Background Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown. Objective To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes. Methods We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4–6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs). Results Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD30). APD50 and APD80 were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (ICa-L) and ~30% reduction (p<0.05) in the transient outward potassium current (ITO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001). Conclusions SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis. PMID:26274906

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture.

PubMed

O'Connell, Ryan P; Musa, Hassan; Gomez, Mario San Martin; Avula, Uma Mahesh; Herron, Todd J; Kalifa, Jerome; Anumonwo, Justus M B

2015-01-01

Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown. To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes. We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4-6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs). Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD30). APD50 and APD80 were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (ICa-L) and ~30% reduction (p<0.05) in the transient outward potassium current (ITO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001). SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

PubMed Central

1975-01-01

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane. PMID:163833

Organic colloids and their influence on low-pressure membrane filtration.

PubMed

Laabs, C; Amy, G; Jekel, M

2004-01-01

Wastewater treatment by low-pressure membrane filtration (MF and UF) is affected to a large extent by macromolecules and colloids. In order to investigate the influence of organic colloids on the membrane filtration process, colloids were isolated from a wastewater treatment plant effluent using a rotary-evaporation pre-concentration step followed by dialysis. Stirred cell tests were carried out using redissolved colloids, with and without additional glass fiber filtration. After constant pressure membrane filtration of 190 L/m2, the initial flux had declined by 50% for colloids > 6-8 kD (glass fiber filtered) with a hydrophilic MF membrane and for colloids > 12-14 kD (glass fiber filtered) with a hydrophobic MF membrane. For the non-filtered colloidal solutions, the flux decline was even steeper with the flux being below 10% of the initial flux after 190 L/m2 were passed through the membranes. As with larger particles, colloids form a filtration cake layer on top of the membrane surface when used as isolates without prior filtration. This filtration cake is easily removed during backwashing. However, polysaccharides as a macromolecular component of the colloid isolate cause severe fouling by the formation of a gel layer on the membrane surface that is difficult to remove completely.

Transfer of a Mycobacterium tuberculosis Genotyping Method, Spoligotyping, from a Reverse Line-Blot Hybridization, Membrane-Based Assay to the Luminex Multianalyte Profiling System

PubMed Central

Cowan, Lauren S.; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T.

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay. PMID:14715809

Chloride Fluxes in Isolated Dialyzed Barnacle Muscle Fibers

PubMed Central

DiPolo, R.

1972-01-01

Chloride outflux and influx has been studied in single isolated muscle fibers from the giant barnacle under constant internal composition by means of a dialysis perfusion technique. Membrane potential was continually recorded. The chloride outfluxes and influxes were 143 and 144 pmoles/cm2-sec (mean resting potential: 58 mv, temperature: 22°–24°C) with internal and external chloride concentrations of 30 and 541 mM, respectively. The chloride conductance calculated from tracer measurements using constant field assumptions is about fourfold greater than that calculated from published electrical data. Replacing 97% of the external chloride ions by propionate reduces the chloride efflux by 51%. Nitrate ions applied either to the internal or external surface of the membrane slows the chloride efflux. The external pH dependence of the chloride efflux follows the external pH dependence of the membrane conductance, in the range pH 3.9–4.7, increasing with decreasing pH. In the range pH 5–9, the chloride efflux increased with increasing pH, in a manner similar to that observed in frog muscle fibers. The titration curve for internal pH changes in the range 4.0–7.0 was quantitatively much different from that for external pH change, indicating significant asymmetry in the internal and external pH dependence of the chloride efflux. PMID:5074810

Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes.

PubMed

Logez, Christel; Berger, Sylvie; Legros, Céline; Banères, Jean-Louis; Cohen, William; Delagrange, Philippe; Nosjean, Olivier; Boutin, Jean A; Ferry, Gilles; Simonin, Frédéric; Wagner, Renaud

2014-01-01

The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

Membrane-acting bacteriocin purified from a soil isolate Pediococcus pentosaceus LB44 shows broad host-range.

PubMed

Kaur, Ramanjeet; Tiwari, Santosh Kumar

2018-04-15

Bacteriocin LB44 was purified from cell-free supernatant (CFS) of Pediococcus pentosaceus LB44 using activity-guided chromatography techniques. It was stable up to 121 °C, pH 2.0-6.0, sensitive to proteinase K, papain and trypsin, and retained complete activity in the presence of organic solvents tested. The molecular weight of bacteriocin was ∼6 kDa and initial ten amino acid residues (GECGMCXECG) suggested a new compound. The loss in viable cell count and K + ion efflux of target cells of Micrococcus luteus suggested bactericidal activity. The cell membrane of bacteriocin-treated cells was found to be ruptured which was further confirmed by Fourier Transform Infrared (FTIR) analysis suggesting interaction of bacteriocin with phospholipids in cell membrane. It showed broad host-range and inhibited the growth of Lactobacillus delbrueckii NRRL B-4525, L. plantarum NRRL B-4496, L. acidophilus NRRL B-4495, Enterococcus hirae LD3, Weissella confusa LM85, Staphylococcus aureus, Salmonella typhi ATCC 13311, Serratia marcescens ATCC 27137, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 29905, Haloferax larsenii HA1, HA3, HA8, HA9 and HA10. These properties suggested a new bacteriocin from soil isolate P. pentosaceus LB44 which may offers possible applications in food-safety and therapeutics. Copyright © 2018 Elsevier Inc. All rights reserved.

Antiviral active peptide from oyster

NASA Astrophysics Data System (ADS)

Zeng, Mingyong; Cui, Wenxuan; Zhao, Yuanhui; Liu, Zunying; Dong, Shiyuan; Guo, Yao

2008-08-01

An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster ( Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10 5 kDa, 5 1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10 5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

PubMed

Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

2013-06-01

Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

Proteins on exocytic vesicles mediate calcium-triggered fusion.

PubMed Central

Vogel, S S; Zimmerberg, J

1992-01-01

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous. Images PMID:1584814

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.

[Effect of nitric oxide on the somatic membrane of rat DRG neurons].

PubMed

Cheng, Hong-Ju; Ma, Ke-Tao; Zhao, Lei; Li, Li; Cao, Ying-Ying; Si, Jun-Qiang

2009-11-01

To observe the role of nitric oxide in dorsal root ganglion (DRG) neurons and its related ionic mechanisms, and explore the function of NO in pain transmission process. In freshly isolated rat DRG samples, using intracellular recording technique, we perfused sodium nitroprusside (NO donor) to observe the role of NO in DRG neurons. In 77.45% of the bath cells, application of sodium nitroprusside (10 -100 mmol/L) induced concentration-dependent membrane hyperpolarization (79/102), and remaining neurons had no response. The membrane conductance increased from control value of (21.06 +/- 1.94) nS to (23.08 +/- 0.92) nS during sodium nitroprusside induced hyperpolarization. L-NAME (1 mmol/L), CdCl2 (0.1 mmol/L) and non-sodium BSS failed to change the amplitude of sodium nitroprusside induced hyperpolarization. When BSS containing 10 mmol/L TEA was used, sodium nitroprusside induced hyperpolarization was obviously inhibited. Sodium nitroprusside could cause concentration-dependent hyperpolarization in DRG neurons by activating K+ channels.

Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure.

PubMed

Szymański, Jędrzej; Janikiewicz, Justyna; Michalska, Bernadeta; Patalas-Krawczyk, Paulina; Perrone, Mariasole; Ziółkowski, Wiesław; Duszyński, Jerzy; Pinton, Paolo; Dobrzyń, Agnieszka; Więckowski, Mariusz R

2017-07-20

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca 2 + handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.

Nanogram calorimetry using microscale suspended SiN{sub x} platforms fabricated via focused ion beam patterning

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

Wickey, K. J.; Chilcote, M.; Johnston-Halperin, E.

2015-01-15

Comprehensive characterization of thermal properties in nanoscale heterostructures requires microscale thermally isolated platforms combined with sensitive thermometry in order to measure small heat accumulations. Amorphous SiN{sub x} membranes are often used for these measurements due to their low thermal conductivity and compatibility with standard fabrication techniques. The total thermal conductance of such SiN{sub x} membranes is typically microwatts per kelvin or higher. Here, we further reduce this thermal coupling to 120 nW/K by using a focused ion beam (FIB) to remove large portions of commercially available amorphous SiN{sub x} membranes, leaving a 100 μm × 100 μm square platform suspendedmore » by 10 μm wide by 325 μm long support legs. We demonstrate the capability of these platforms by measuring the heat capacity of a 6.2 ng Au sample and show that it matches well with established specific heat of bulk Au.« less

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 prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.

Cholinergically-induced changes in outward currents in hair cells isolated from the semicircular canal of the frog.

PubMed

Housley, G D; Norris, C H; Guth, P S

1990-01-01

Two cholinergically-induced modulations of membrane conductances have been identified in hair cells isolated from the crista ampullaris of the leopard frog (Rana pipiens), using the whole cell recording configuration of the patch clamp technique. Of 56 crista hair cells tested, 28 showed drug-induced changes in membrane current or membrane potential which were repeatable and could be reversed with washout of drug. The predominant effect (observed in 20 hair cells) of acetylcholine (Ach, 100 microM) to 1mM) or carbachol (1 microM to 50 microM) applied to these hair cells was the reduction of an outward current corresponding to a change in conductance of approximately -0.22 nS. This action by Ach on hair cells has been inferred from previous studies of afferent fiber discharge which reported an increase in firing rate with stimulation of efferent fibers or exogenous application of cholinomimetics (Bernard et al., 1985; Valli et al., 1986; Guth et al., 1986; Norris et al., 1988a). The Ach-induced reduction in outward current was associated with a depolarization of the zero-current membrane potential by approximately +2.5 mV. In a total of 8 hair cells, an Ach-induced reversible increase in outward current was recorded. Changes in conductance were approximately +0.13 nS and were associated with a hyperpolarization of the zero-current membrane potential by approximately -2.2 mV. This current increase is likely to be responsible for the inhibitory post-synaptic potentials (IPSPs) which have previously been recorded intracellularly from acoustico-lateralis hair cells during stimulation of the efferent innervation (Flock and Russell, 1976; Ashmore and Russell, 1982; Art et al., 1984, 1985). Of the remaining 28 hair cells, six cells failed to exhibit any change in membrane conductance or membrane potential in the presence of cholinomimetics while an additional 15 cells exhibited decreases, and 7 cells exhibited increases in outward conductance, during application of Ach or carbachol, which were neither reversible with washout nor repeatable. The Ach-induced decrease in outward current could be reversible blocked by removal of Ca2+ from the external solution. The antagonism of the Ach-induced decrease in outward current by atropine (10(-5) M) suggests that this current may correspond to a facilitatory, 'atropine-preferring' Ach receptor mediated response previously identified in the isolated semicircular canal (Norris et al., 1988a).(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of human mesenchymal stromal cells from four neonatal tissues: Amniotic membrane, chorionic membrane, placental decidua and umbilical cord.

PubMed

Araújo, Anelise Bergmann; Salton, Gabrielle Dias; Furlan, Juliana Monteiro; Schneider, Natália; Angeli, Melissa Helena; Laureano, Álvaro Macedo; Silla, Lúcia; Passos, Eduardo Pandolfi; Paz, Ana Helena

2017-05-01

Mesenchymal stromal cells (MSCs) are being investigated as a potential alternative for cellular therapy. This study was designed to compare the biological characteristics of MSCs isolated from amniotic membrane (A-MSCs), chorionic membrane (C-MSCs), placental decidua (D-MSCs) and umbilical cord (UC-MSCs) to ascertain whether any one of these sources is superior to the others for cellular therapy purposes. MSCs were isolated from amniotic membrane, chorionic membrane, umbilical cord and placental decidua. Immunophenotype, differentiation ability, cell size, cell complexity, polarity index and growth kinetics of MSCs isolated from these four sources were analyzed. MSCs were successfully isolated from all four sources. Surface marker profile and differentiation ability were consistent with human MSCs. C-MSCs in suspension were the smallest cells, whereas UC-MSCs presented the greatest length and least width. A-MSCs had the lowest polarity index and UC-MSCs, as more elongated cells, the highest. C-MSCs, D-MSCs and UC-MSCs exhibited similar growth capacity until passage 8 (P8); C-MSCs presented better lifespan, whereas insignificant proliferation was observed in A-MSCs. Neonatal and maternal tissues can serve as sources of multipotent stem cells. Some characteristics of MSCs obtained from four neonatal tissues were compared and differences were observed. Amniotic membrane was the least useful source of MSCs, whereas chorionic membrane and umbilical cord were considered good options for future use in cell therapy because of the known advantages of immature cells. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts.

PubMed Central

Schmidt, H; Heinz, E

1990-01-01

Envelope membranes isolated from chloroplasts of spinach (Spinacia oleracea) desaturate oleoyl groups in monogalactosyl diacylglycerol to linoleoyl groups. The desaturation requires NADPH in combination with ferredoxin and is not restricted to monogalactosyl diacylglycerol, since it is also observed in biosynthetic intermediates as, for example, in phosphatidic acid. This indicates a certain degree of unspecificity of the oleate desaturase in isolated envelope membranes. Lipid desaturation is another important function of chloroplast envelopes. PMID:11607123

Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins.

PubMed

Chaney, L K; Jacobson, B S

1983-08-25

Plasma membrane (PM) can be isolated by binding to a positively charged solid support. Using this concept, we have developed a novel method of PM isolation using cationic colloidal silica. The method is designed for the comparative study of various physiological states of PM and for transbilayer protein mapping. The procedure consists of coating intact cells with a dense pellicle of silica particles and polyanion. Since cells remain intact during pellicle formation, the external face of the PM is selectively coated. The pellicle greatly enhances PM density and stabilizes it against vesiculation or lateral reorientation. Upon cell lysis, large open sheets of PM are rapidly isolated by centrifugation. PM from Dictyostelium discoideum was prepared by this method. Marker enzymes, cell surface labeling and microscopy demonstrate that the PM was isolated in high yield (70-80%) with a 10-17-fold purification and only low levels of cytoplasmic contamination. The pellicle remains intact during cell lysis and membrane isolation, shielding the external surface of the membranes up to 92% from chemical or enzymatic attack. The PM can thus be labeled selectively from inside and/or outside. Transmembrane proteins were identified in Dictyostelium PM by means of lactoperoxidase iodination and autoradiography.

Voltage clamp methods for the study of membrane currents and SR Ca2+ release in adult skeletal muscle fibres

PubMed Central

Hernández-Ochoa, Erick O.; Schneider, Martin F.

2012-01-01

Skeletal muscle excitation-contraction (E-C)1 coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)2 Ca2+ release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca2+, due to depolarization-initiated SR Ca2+ release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or ‘high resistance gap’ techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca2+ signalling properties of mouse skeletal muscle membranes are being investigated. PMID:22306655

Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process.

PubMed Central

Stremmel, W

1988-01-01

The mechanism by which fatty acids enter cardiomyocytes is unclear. Therefore, the influx kinetics of [3H]oleate into isolated rat heart myocytes were examined. Cells were incubated at 37 degrees C with [3H]oleate bound to albumin in various molar ratios and the initial rate of uptake (V0) was determined as a function of the unbound oleate concentration in the medium. V0 was saturable with increasing oleate concentrations incubated (Km 78 nM; Vmax 1.9 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Furthermore, binding of [3H]oleate to isolated plasma membranes of cardiomyocytes was saturable, revealing a KD of 42 nM, and was inhibited by heat denaturation or trypsin pretreatment of the membranes. From these membranes a single 40-kD protein with high affinity for a variety of long chain fatty acids was isolated. With a monospecific antibody to this membrane protein, binding as well as cellular influx of [3H]oleate was selectively inhibited. These data indicate that at least a portion of myocardial fatty acid uptake is mediated by a specific membrane protein. Images PMID:3343344

Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

PubMed

Gerstle, Zoe; Desai, Rohan; Veatch, Sarah L

2018-01-01

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy. © 2018 Elsevier Inc. All rights reserved.

Water hyacinth cellulose-based membrane for adsorption of liquid waste dyes and chromium

NASA Astrophysics Data System (ADS)

Agtasia Putri, Cintia; Yulianti, Ian; Desianna, Ika; Sholihah, Anisa; Sujarwata

2018-04-01

Water hyacinth (Eichornia crassipes) is a weed in aquatic area whose trunk contains a lot of cellulose. Cellulose contained can be used as dyes adsorbent in a form of composite membrane. This study aims to investigate the capacity of water hyacinth cellulose-based membrane to adsorb dye and Chromium (Cr) contained in liquid. The process of membrane fabrication begins with isolation of water hyacinth cellulose. The isolated cellulose powder was used to make the membrane by mixing it with polyvinyl alcohol-polyethylene glycol (PVA-PEG) with various compositions. The morphology of membrane surface was analyzed using CCD microscope. The analysis using Ultraviolet Visible Spectroscopy (UV-Vis) and Atomic Absorption Spectroscopy (AAS) indicate that the membrane with composition ratio of cellulose: PVA: PEG of 6.5: 2.5: 1 adsorb Cr up to 38.75%.

Foam metal metamaterial panel for mechanical waves isolation

NASA Astrophysics Data System (ADS)

Hua, Lei; Sun, Hongwei; Gu, Jinliang

2016-04-01

This paper presents modeling, analysis techniques and experiment of foam metal metamaterial panel for Broadband Vibration Absorption. For a unit cell of an infinite foam metal metamaterial panel, governing equations are derived using the extended Hamilton principle. The concepts of negative effective mass and stiffness and how the spring-mass-damper subsystems create a stopband are explained in detail. Numerical simulations reveal that the actual working mechanism of the proposed metamaterial panel is based on the concept of conventional mechanical vibration absorbers. It uses the incoming elastic wave in the panel to resonate the integrated membrane-mass-damper absorbers to vibrate in their optical mode at frequencies close to but above their local resonance frequencies to create shear forces and bending moments to straighten the panel and stop the wave propagation. Moreover, a two-dimension acoustic foam metal metamaterial panel consisting of lumped mass and elastic membrane is proposed in the lab. We do experiments on the model and The results validate the concept and show that, for two-dimension acoustic foam metal metamaterial panel do exist two vibration modes. For the wave absorption, the mass of each cell should be considered in the design. With appropriate design calculations, the proposed two-dimension acoustic foam metal metamaterial panel can be used for absorption of low-frequency waves and hence expensive micro-manufacturing techniques are not needed for design and manufacturing of such foam metal metamaterial panel for low-frequency waves absorption/isolation.

Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment

PubMed Central

Waldenström, Jonas; Axelsson-Olsson, Diana; Olsen, Björn; Hasselquist, Dennis; Griekspoor, Petra; Jansson, Lena; Teneberg, Susann; Svensson, Lovisa; Ellström, Patrik

2010-01-01

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations. PMID:20140204

Properties of the cromakalim-induced potassium conductance in smooth muscle cells isolated from the rabbit portal vein.

PubMed Central

Beech, D. J.; Bolton, T. B.

1989-01-01

1. Single smooth muscle cells were isolated freshly from the rabbit portal vein and membrane currents were recorded by the whole-cell or excised patch configurations of the patch-clamp technique at room temperature. 2. Cromakalim (Ckm, 10 microM) induced a potassium current (ICkm) that showed no pronounced voltage-dependence and had low current noise. 3. This current, ICkm, was inhibited by (in order of potency): phencyclidine greater than quinidine greater than 4-aminopyridine greater than tetraethylammonium ions (TEA). These drugs inhibited the delayed rectifier current, IdK, which is activated by depolarization of the cell, with the same order of potency. 4. Large conductance calcium-activated potassium channels (LKCa) in isolated membrane patches were blocked by (in order of potency) quinidine greater than TEA approximately phencyclidine. 4-Aminopyridine was ineffective. A similar order of potency was found for block of spontaneous transient outward currents thought to represent bursts of openings of LKCa channels. 5. The low current noise of ICkm at positive potentials, and its susceptibility to inhibitors indicated that it was not carried by LKCa channels, and that it may be carried by channels which underlie IdK. It was observed that when ICkm was activated, IdK was reduced. However, in two experiments, ICkm was much more susceptible to glibenclamide than IdK; possible reasons for this are discussed. PMID:2590772

Ectopic expression of plasma membrane targeted subunits of the Ndc80-complex as a tool to study kinetochore biochemistry.

PubMed

Holmström, Tim H; Rehnberg, Jonathan; Ahonen, Leena J; Kallio, Marko J

2009-06-01

Genomic stability depends on the normal function of the kinetochore, a multi-protein assemblage, which consists of over 80 molecules including both constitutive and transiently binding components. Information regarding the spatial-temporal assembly of kinetochore subcomplexes is often limited by technical difficulties in their isolation. To study kinetochore subcomplex formation, we targeted separately Hec1 and Spc24, two subunits of the Ndc80 kinetochore compilation, to the plasma membrane by fusing them with the amino-terminal palmitoylation and myristoylation (pm) sequence of the receptor tyrosine kinase Fyn. We found that in early mitotic cells, pm-GFP-Hec1 and pm-GFP-Spc24 fusion proteins localised to the plasma membrane and were able to recruit all subunits of the Ndc80 complex (Ndc80/Hec1, Nuf2, Spc24 and Spc25) to these foci. In interphase cells, only Hec1-Nuf2 and Spc24-Spc25 heterodimers accumulated to the plasma membrane foci. The results propose that the assembly of Ndc80 tetramer can take place outside of the kinetochore but requires co-factors that are only present in mitotic cells. These findings provide the first experimental evidence on the successful employment of the plasma membrane targeting technique in the study of kinetochore biochemistry.

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.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

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

Pardo-Andreu, Gilberto L., E-mail: gilbertopardo@infomed.sld.cu; Departamento de Fisica e Quimica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. Cafe s/n, 14040-903 Ribeirao Preto, SP; Nunez-Figueredo, Yanier

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 {mu}M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca{sup 2+} efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. Allmore » effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP{sup +} transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. - Graphical abstract: Guttiferone-A permeabilizes mitochondrial membrane and induces cancer cell death Display Omitted Highlights: > We addressed the involvement of mitochondria in guttiferone (GA) toxicity toward cancer cells. > GA promoted membrane permeabilization, membrane potential dissipation, NAD(P)H depletion, ROS accumulation and ATP depletion. > These actions could be implicated in the well-documented anti-cancer property of GA/structure related compounds.« less

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

PubMed Central

Green, Anita A.; Newell, Peter C.

1974-01-01

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N2 and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [125I]iodide. 5′-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH–cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH–cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants. ImagesPLATE 1 PMID:4156170

Development of an optimal filter substrate for the identification of small microplastic particles in food by micro-Raman spectroscopy.

PubMed

Oßmann, Barbara E; Sarau, George; Schmitt, Sebastian W; Holtmannspötter, Heinrich; Christiansen, Silke H; Dicke, Wilhelm

2017-06-01

When analysing microplastics in food, due to toxicological reasons it is important to achieve clear identification of particles down to a size of at least 1 μm. One reliable, optical analytical technique allowing this is micro-Raman spectroscopy. After isolation of particles via filtration, analysis is typically performed directly on the filter surface. In order to obtain high qualitative Raman spectra, the material of the membrane filters should not show any interference in terms of background and Raman signals during spectrum acquisition. To facilitate the usage of automatic particle detection, membrane filters should also show specific optical properties. In this work, beside eight different, commercially available membrane filters, three newly designed metal-coated polycarbonate membrane filters were tested to fulfil these requirements. We found that aluminium-coated polycarbonate membrane filters had ideal characteristics as a substrate for micro-Raman spectroscopy. Its spectrum shows no or minimal interference with particle spectra, depending on the laser wavelength. Furthermore, automatic particle detection can be applied when analysing the filter surface under dark-field illumination. With this new membrane filter, analytics free of interference of microplastics down to a size of 1 μm becomes possible. Thus, an important size class of these contaminants can now be visualized and spectrally identified. Graphical abstract A newly developed aluminium coated polycarbonate membrane filter enables automatic particle detection and generation of high qualitative Raman spectra allowing identification of small microplastics.

Double-membrane triple-electrolyte redox flow battery design

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

Yushan, Yan; Gu, Shuang; Gong, Ke

A redox flow battery is provided having a double-membrane (one cation exchange membrane and one anion exchange membrane), triple-electrolyte (one electrolyte in contact with the negative electrode, one electrolyte in contact with the positive electrode, and one electrolyte positioned between and in contact with the two membranes). The cation exchange membrane is used to separate the negative or positive electrolyte and the middle electrolyte, and the anion exchange membrane is used to separate the middle electrolyte and the positive or negative electrolyte. This design physically isolates, but ionically connects, the negative electrolyte and positive electrolyte. The physical isolation offers greatmore » freedom in choosing redox pairs in the negative electrolyte and positive electrolyte, making high voltage of redox flow batteries possible. The ionic conduction drastically reduces the overall ionic crossover between negative electrolyte and positive one, leading to high columbic efficiency.« less

Fabrication and Performance of Large Format Transition Edge Sensor Microcalorimeter Arrays

NASA Technical Reports Server (NTRS)

Chervenak, James A.; Adams, James S.; Bandler, Simon R.; Busch, Sara E.; Eckart, M. E.; Ewin, A. E.; Finkbeiner, F. M.; Kilbourne, C. A.; Kelley, R. L.; Porst, Jan-Patrick;

Occurrence of nontuberculous mycobacteria in environmental samples.

PubMed

Covert, T C; Rodgers, M R; Reyes, A L; Stelma, G N

1999-06-01

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

PubMed

Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

2016-01-01

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Enhanced synergism of antibiotics with zinc oxide nanoparticles against extended spectrum β-lactamase producers implicated in urinary tract infections

NASA Astrophysics Data System (ADS)

Bhande, Rashmi M.; Khobragade, C. N.; Mane, R. S.; Bhande, S.

2013-01-01

In this study, enhanced synergistic bioactivity of zinc oxide nanoparticles (ZnO NPs) with β-lactam antibiotics were evaluated against a panel of clinically isolated extended spectrum β-lactamase producers implicated in urinary tract infections. Chemically synthesized zinc oxide nanoparticles (15 nm) were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), high resolution transmittance electron microscopy (HR-TEM), selective area electron diffraction (SAED), X-ray photoelectron spectroscopy (XPS), and UV-Visible spectrophotometry techniques. The antimicrobial potency (10 ± 0.66, 12, 11.33 ± 1.10, and 0.7 ± 0.66 mm inhibiting zone) and minimum inhibitory concentrations (80, 60, 30, 50 μg/ml) of ZnO NPs were tested separately whereas time-kill and membrane leakage assays were evaluated in combination with ZnO NPs+ cefotaxime, ampicillin, ceftriaxone, cefepime against the β-lactamase producer strains of E. coli, K. pneumoniae, S. paucimobilis, and P. aeruginosa, respectively. Time-kill curve dynamics of ZnO NPs with β-lactam antibiotics revealed enhanced bactericidal activity (50, 85, 58, 50 % fold inhibition) by delaying the exponential and stationary phases of all isolates when tested separately. Posttime-kill effect was studied on cell membrane by assaying leakage of reducing sugars (130.2, 124.7, 137, and 115.8 μg/bacterial dry weight of 1 mg (μg/mg) and proteins (15, 10, 16, 18 μg/mg). These assays revealed that membrane leakage was due to synergism of ZnO NPs+ β-lactam antibiotics which successfully damage cell membrane thereby leading to death of all ESBL producers. The results demonstrate the utilization of ZnO NPs as a potentiator of β-lactam antibiotics and suggest the possibility to use nanoparticles in a combination therapy to treat UTI.

Anti-methicillin-resistance Staphylococcus aureus (MRSA) compounds from Bauhinia kockiana Korth. And their mechanism of antibacterial activity.

PubMed

Chew, Yik Ling; Mahadi, Adlina Maisarah; Wong, Kak Ming; Goh, Joo Kheng

2018-02-20

Bauhinia kockiana originates from Peninsular Malaysia and it is grown as a garden ornamental plant. Our previous study reported that this plant exhibited fairly strong antioxidant and antimicrobial activities. This paper focused on the assessment of the antibacterial activity of B. kockiana towards methicillin-resistance Staphylococcus aureus (MRSA), to purify and to identify the antibacterial compounds, and to determine the mechanism of antibacterial activity. Antibacterial activity of B. kockiana flower was evaluated qualitatively and quantitatively using disc diffusion assay and microbroth dilution method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extracts were examined. Phytochemical analysis was performed to determine the classes of phytochemicals in the extracts. Bioactivity guided isolation was employed to purify the antibacterial agents and identified via various spectroscopy methods. Scanning electron microscopy (SEM) technique was used to evaluate the antibacterial mechanism of extract and compounds isolated. B. kockiana flower was found to exhibit fairly strong antibacterial activity towards both strains of MRSA bacteria used, MIC varies from 62.5-250 μg/mL. Tannins and flavonoids have been detected in the phytochemical analysis. Gallic acid and its ester derivatives purified from ethyl acetate extract could inhibit MRSA at 250-500 μg/mL. SEM revealed that the cells have undergone plasmolysis upon treatment with the extract and compounds. Tannins and polyphenols are the antibacterial components towards MRSA in B. kockiana. Massive leakage of the cell content observed in treated cells showed that the phytochemicals have changed the properties of the cell membranes. Amphiphilic nature of the compounds exhibited the antibacterial activity towards MRSA via three stages: (1) cell membrane attachment; (2) cell membrane fluidity modification; and (3) cell membrane structure disruption.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis.

PubMed

Wilson, Kris; Webster, Scott P; Iredale, John P; Zheng, Xiaozhong; Homer, Natalie Z; Pham, Nhan T; Auer, Manfred; Mole, Damian J

2017-12-15

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

NASA Astrophysics Data System (ADS)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

A method for detergent-free isolation of membrane proteins in their local lipid environment.

PubMed

Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow, Rosemary A; Lin, Yu-Pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench, Stephen P; Dafforn, Timothy R

2016-07-01

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Comprehensive isolation of natural organic matter from water for spectral characterizations and reactivity testing

USGS Publications Warehouse

Leenheer, J.A.; Croue, J.-P.; Benjamin, M.; Korshin, G.V.; Hwang, C.J.; Bruchet, A.; Aiken, G.R.

2000-01-01

A variety of approaches were tested to comprehensively isolate natural organic matter (NOM) from water. For waters with high NOM concentrations such as the Suwannee River, Georgia, approaches that used combinations of membrane concentrations, evaporative concentrations, and adsorption on nonionic XAD resins, ion exchange resins and iron oxide coated sand isolated over 90% of the NOM. However, for waters with low NOM concentrations, losses of half of the NOM were common and desalting of NOM isolates was a problem. A new comprehensive approach was devised and tested on the Seine River, France in which 100 L of filtered water was sodium softened by ion exchange and vacuum evaporated to 100 mL. Colloids (32% of the NOM) were isolated using a 3,500 Dalton membrane by dialysis against 0.1 M HCl and 0.2 M HF to remove salts and silica. On the membrane permeate, hydrophobic NOM (42%) was isolated using XAD-8 resin and hydrophilic NOM (26%) was isolated using a variety of selective desalting precipitations. The colloid fraction was characterized by IR and NMR spectroscopy as N-acetylamino sugars. ?? 2000 American Chemical Society.

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

PubMed

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

1987-11-01

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

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

PubMed

Verméglio, André; Lavergne, Jérôme; Rappaport, Fabrice

2016-01-01

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

PubMed Central

Logez, Christel; Berger, Sylvie; Legros, Céline; Banères, Jean-Louis; Cohen, William; Delagrange, Philippe; Nosjean, Olivier; Boutin, Jean A.; Ferry, Gilles

2014-01-01

The human melatonin MT1 receptor—belonging to the large family of G protein-coupled receptors (GPCRs)—plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches. PMID:24959712

Human Lung Small Airway-on-a-Chip Protocol.

PubMed

Benam, Kambez H; Mazur, Marc; Choe, Youngjae; Ferrante, Thomas C; Novak, Richard; Ingber, Donald E

2017-01-01

Organs-on-chips are microfluidic cell culture devices created using microchip manufacturing techniques that contain hollow microchannels lined by living cells, which recreate specialized tissue-tissue interfaces, physical microenvironments, and vascular perfusion necessary to recapitulate organ-level physiology in vitro. Here we describe a protocol for fabrication, culture, and operation of a human lung "small airway-on-a-chip," which contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an underlying microvascular endothelium that experiences fluid flow. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin rigid porous membrane; this requires less than 1 day to complete. Next, primary human airway bronchiolar epithelial cells isolated from healthy normal donors or patients with respiratory disease are cultured on the porous membrane within one microchannel while lung microvascular endothelial cells are cultured on the opposite side of the same membrane in the second channel to create a mucociliated epithelium-endothelium interface; this process take about 4-6 weeks to complete. Finally, culture medium containing neutrophils isolated from fresh whole human blood are flowed through the microvascular channel of the device to enable real-time analysis of capture and recruitment of circulating leukocytes by endothelium under physiological shear; this step requires less than 1 day to complete. The small airway-on-a-chip represents a new microfluidic tool to model complex and dynamic inflammatory responses of healthy and diseased lungs in vitro.

A survey of membrane oxygenator heat-exchanger integrity testing at cardiac surgery centres in Great Britain and Ireland.

PubMed

Carlton, Matthew; Campbell, John

2013-11-01

Membrane oxygenator heat exchanger (HE) device failure is reported to be very low for both short- and long-term extracorporeal devices. All oxygenator manufacturers provide instructions for leak testing of their HE devices prior to patient use. In addition to these recommendations, since 2006 at Nottingham University Hospitals (NUH) we have also additionally pressure tested HE devices prior to use. We conducted a national survey of cardiac centers in Great Britain and Ireland to determine the methods undertaken in individual centers for validation of the integrity of HE devices. Furthermore, we also collected information on the routine maintenance techniques utilized within these centers to inhibit microbial growth in the water used in the heater-cooler units (HCUs). In total, 34 responses were collected from the 57 centers performing cardiac surgery, producing a response rate of 60%. Of the responding centers, 71% are adhering to manufacturer's recommended guidelines of circulating the water through the device for 5 minutes. Of these centers, 17% reported detecting a leak between the HE and membrane compartment of the oxygenator. In responding centers, 29% reported using the pressure test technique. In the centers utilizing pressure testing, 60% reported detecting a leak. This survey reports an association of a greater HE leak detection rate using the pressure test technique compared to using water testing in isolation (p = 0.034). We believe the pressure testing method provides the perfusionist with confidence in the integrity of the HE for short- and long-term circulatory support devices prior to use in both elective and emergency situations.

Linking ceragenins to water-treatment membranes to minimize biofouling.

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

Hibbs, Michael R.; Altman, Susan Jeanne; Feng, Yanshu

Ceragenins were used to create biofouling resistant water-treatment membranes. Ceragenins are synthetically produced antimicrobial peptide mimics that display broad-spectrum bactericidal activity. While ceragenins have been used on bio-medical devices, use of ceragenins on water-treatment membranes is novel. Biofouling impacts membrane separation processes for many industrial applications such as desalination, waste-water treatment, oil and gas extraction, and power generation. Biofouling results in a loss of permeate flux and increase in energy use. Creation of biofouling resistant membranes will assist in creation of clean water with lower energy usage and energy with lower water usage. Five methods of attaching three different cerageninmore » molecules were conducted and tested. Biofouling reduction was observed in the majority of the tests, indicating the ceragenins are a viable solution to biofouling on water treatment membranes. Silane direct attachment appears to be the most promising attachment method if a high concentration of CSA-121a is used. Additional refinement of the attachment methods are needed in order to achieve our goal of several log-reduction in biofilm cell density without impacting the membrane flux. Concurrently, biofilm forming bacteria were isolated from source waters relevant for water treatment: wastewater, agricultural drainage, river water, seawater, and brackish groundwater. These isolates can be used for future testing of methods to control biofouling. Once isolated, the ability of the isolates to grow biofilms was tested with high-throughput multiwell methods. Based on these tests, the following species were selected for further testing in tube reactors and CDC reactors: Pseudomonas ssp. (wastewater, agricultural drainage, and Colorado River water), Nocardia coeliaca or Rhodococcus spp. (wastewater), Pseudomonas fluorescens and Hydrogenophaga palleronii (agricultural drainage), Sulfitobacter donghicola, Rhodococcus fascians, Rhodobacter katedanii, and Paracoccus marcusii (seawater), and Sphingopyxis spp. (groundwater). The testing demonstrated the ability of these isolates to be used for biofouling control testing under laboratory conditions. Biofilm forming bacteria were obtained from all the source water samples.« less

Series resistance compensation for whole-cell patch-clamp studies using a membrane state estimator

PubMed Central

Sherman, AJ; Shrier, A; Cooper, E

1999-01-01

Whole-cell patch-clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the series resistance (R(s)) of the recording pipette limits the bandwidth of the whole-cell configuration, making it difficult to measure rapid ionic currents. To increase bandwidth, it is necessary to compensate for R(s). Most methods of R(s) compensation become unstable at high bandwidth, making them hard to use. We describe a novel method of R(s) compensation that overcomes the stability limitations of standard designs. This method uses a state estimator, implemented with analog computation, to compute the membrane potential, V(m), which is then used in a feedback loop to implement a voltage clamp; we refer to this as state estimator R(s) compensation. To demonstrate the utility of this approach, we built an amplifier incorporating state estimator R(s) compensation. In benchtop tests, our amplifier showed significantly higher bandwidths and improved stability when compared with a commercially available amplifier. We demonstrated that state estimator R(s) compensation works well in practice by recording voltage-gated Na(+) currents under voltage-clamp conditions from dissociated neonatal rat sympathetic neurons. We conclude that state estimator R(s) compensation should make it easier to measure large rapid ionic currents with whole-cell patch-clamp techniques. PMID:10545359

Low-stress photosensitive polyimide suspended membrane for improved thermal isolation performance

NASA Astrophysics Data System (ADS)

Fan, J.; Xing, R. Y.; Wu, W. J.; Liu, H. F.; Liu, J. Q.; Tu, L. C.

2017-11-01

In this paper, we introduce a method of isolating thermal conduction from silicon substrate for accommodating thermal-sensitive micro-devices. This method lies in fabrication of a low-stress photosensitive polyimide (PSPI) suspension structure which has lower thermal conductivity than silicon. First, a PSPI layer was patterned on a silicon wafer and hard baked. Then, a cavity was etched from the backside of the silicon substrate to form a membrane or a bridge-shape PSPI structure. After releasing, a slight deformation of about 20 nm was observed in the suspended structures, suggesting ultralow residual stress which is essential for accommodating micro-devices. In order to investigate the thermal isolation performance of the suspended PSPI structures, micro Pirani vacuum gauges, which are thermal-sensitive, had been fabricated on the PSPI structures. The measurement results illustrated that the Pirani gauges worked as expected in the range from 1- 470 Pa. Moreover, the results of the Pirani gauges based on the membrane and bridge structures were comparable, indicating that the commonly used bridge-shape structure for further reducing thermal conduction was unnecessary. Due to the excellent thermal isolation performance of PSPI, the suspended PSPI membrane is promising to be an outstanding candidate for thermal isolation applications.

Isolation of Hybridomas for Golgi-associated Proteins and a Plant Calmodulin

NASA Technical Reports Server (NTRS)

Kuzmanoff, K. M.; Ray, P. M.

1985-01-01

The demonstration of a role for calcium in the mechanism of the gravitropic response indicates a role for calmodulin. Localization studies indicate that plant cell walls have a high content of calmodulin which suggests a regulatory role for CaM in both gravitropic curvature and auxin-induced growth. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the ctivity of Golgi-localized B-1,4-glucan synthase (GS), an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. In order to determine whether auxin stimulates GS activity either by modulation of existing enzyme or induces de novo formation of Golgi glucan synthase, a study was undertaken to isolate and quantitate glucan synthase. This enzyme appears to be an integral protein of the Golgi membrane and has resisted isolation with retention of activity. The production of monoclonal antibody for glucan synthase was undertaken due to the inability to isolate GS by standard detergent/liposome techniques.

The Novelty in Fabrication of Poly Vinyl Alcohol/κ-Carrageenan Hydrogel with Lactobacillus bulgaricus Extract as Anti-inflammatory Wound Dressing Agent.

PubMed

El-Fawal, Gomaa F; Yassin, Abdelrahman M; El-Deeb, Nehal M

2017-07-01

Material barrier properties to microbes are an important issue in many pharmaceutical applications like wound dressings. A wide range of biomaterials has been used to manage the chronic inflamed wounds. Eight hydrogel membranes of poly vinyl alcohol (PVA) with κ-carrageenan (KC) and Lactobacillus bulgaricus extract (LAB) have been prepared by using freeze-thawing technique. To evaluate the membranes efficiency as wound dressing agents, various tests have been done like gel fraction, swelling behavior, mechanical properties, etc. The antibacterial activities of the prepared membranes were tested against the antibiotic-resistant bacterial isolates. In addition, the safety usage of the prepared hydrogel was checked on human dermal fibroblast cells. The anti-inflammatory properties of the prepared hydrogel on LPS-PBMC cell inflammatory model were quantified using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-qPCR). The analysis data of TGA, SEM, gel fraction, and swelling behavior showed changes in properties of prepared PVA\\KC\\LAB hydrogel membrane than pure PVA hydrogel membrane. The antibacterial activities of the prepared membranes augmented in LAB extract-prepared membranes. Out of the eight used hydrogel membranes, the PVAKC4 hydrogel membrane is the safest one on fibroblast cellular proliferation with a maximum proliferation percentage 97.3%. Also, all the used hydrogel membrane showed abilities to reduce the concentration of IL-2 and IL-8 compared with both negative and positive control. In addition, almost all the prepared hydrogel membrane showed variable abilities to downregulate the expression of TNF-α gene with superior effect of hydrogel membrane KC1. PVA/KC/LAB extract hydrogel membrane may be a promising material for wound dressing application and could accelerate the healing process of the chronic wound because of its antimicrobial and anti-inflammatory properties.

Developing synthesis techniques for zeolitic-imidazolate framework membranes for high resolution propylene/propane separation

NASA Astrophysics Data System (ADS)

Kwon, Hyuk Taek

Propylene/propane separation is one of the most challenging separations, currently achieved by energy-intensive cryogenic distillation. Despite the great potentials for energy-efficient membrane-based propylene/propane separation processes, no commercial membranes are available due to the limitations (i.e., low selectivity) of current polymeric materials. Zeolitic imidazolate frameworks (ZIFs) are promising membrane materials primarily due to their well-defined ultra-micropores with controllable surface chemistry along with their relatively high thermal/chemical stabilities. In particular, ZIF-8 with the effective aperture size of ~ 4.0 A has been shown very promising for propylene/propane separation. Despite the extensive research on ZIF-8 membranes, only a few of ZIF-8 membranes have displayed good propylene/propane separation performances presumably due to the challenges of controlling the microstructures of polycrystalline membranes. Since the membrane microstructures are greatly influenced by processing techniques, it is critically important to develop new techniques. In this dissertation, three state-of-the-art ZIF membrane synthesis techniques are developed. The first is a one-step in-situ synthesis technique based on the concept of counter diffusion. The technique enabled us to obtain highly propylene selective ZIF-8 membranes in less than a couple of hours with exceptional mechanical strength. Most importantly, due to the nature of the counter-diffusion concept, the new method offered unique opportunities such as healing defective membranes (i.e., poorly-intergrown) as well as significantly reducing the consumption of costly ligands and organic solvents. The second is a microwave-assisted seeding technique. Using this new seeding technique, we were able to prepare seeded supports with a high packing density in a couple of minutes, which subsequently grown into highly propylene-selective ZIF-8 membranes with an average propylene/propane selectivity of ~40. The last is a heteroepitaxial growth technique. The first well-intergrown membranes of ZIF-67 (Co-substituted ZIF-8) by heteroepitaxially growing ZIF-67 on ZIF-8 seed layers were reported. The ZIF-67 membranes exhibited impressively high propylene/propane separation capabilities. The presence of a methanol co-solvent in the growth solution was critically important to reproducibly prepare high quality ZIF-67 membranes. Furthermore, when the tertiary growth of ZIF-8 layers was applied to the ZIF-67 membranes, the membranes exhibited unprecedentedly high propylene/propane separation factors of ~ 200 possibly due to enhanced grain boundary structure.

Visualization of membrane RNAs

PubMed Central

JANAS, TADEUSZ; YARUS, MICHAEL

2003-01-01

Using fluorescence microscopy, we show that previously isolated membrane-binding RNAs coat artificial phospholipid membranes relatively uniformly, except for a frequent tendency to concentrate at bends, membrane junctions, and other unusual sites. Membrane RNAs can also be visualized as single molecules or isolated complexes by atomic force microscopy (AFM) of free RNAs on mica. Finally, RNAs can be seen within membranes by AFM of RNA-liposomes immobilized on hydrophobic mica surfaces. Monomer RNAs appear globular, as expected for small RNAs. When mixed under conditions in which RNAs bind bilayers, RNA 9 and RNA 10 combine to yield about 80% of RNAs as mainly linear oligomers of ≈2–8 molecules. Once inserted in membranes, the RNAs oligomerize further, yielding larger, irregular ropelike structures that prefer the edges of altered lipid patches. These properties can be interpreted in terms of RNA–RNA loop interactions, and the RNA effects on membranes can be explained in terms of an RNA preference for irregular lipid conformations. The RNA-bilayer system poses new opportunities for combining the properties of membranes and RNA in contemporary cells, and also in the ribocytes of an RNA world. PMID:14561885

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale.

PubMed

G Junior, Daniel S; Araújo, Flábio R; Almeida Junior, Nalvo F; Adi, Said S; Cheung, Luciana M; Fragoso, Stenio P; Ramos, Carlos A N; Oliveira, Renato Henrique M de; Santos, Caroline S; Bacanelli, Gisele; Soares, Cleber O; Rosinha, Grácia M S; Fonseca, Adivaldo H

2010-11-01

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Isolation of plasma membrane-associated membranes from rat liver.

PubMed

Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

2014-02-01

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

Isolation, Separation, and Preconcentration of Biologically Active Compounds from Plant Matrices by Extraction Techniques.

PubMed

Raks, Victoria; Al-Suod, Hossam; Buszewski, Bogusław

2018-01-01

Development of efficient methods for isolation and separation of biologically active compounds remains an important challenge for researchers. Designing systems such as organomineral composite materials that allow extraction of a wide range of biologically active compounds, acting as broad-utility solid-phase extraction agents, remains an important and necessary task. Selective sorbents can be easily used for highly selective and reliable extraction of specific components present in complex matrices. Herein, state-of-the-art approaches for selective isolation, preconcentration, and separation of biologically active compounds from a range of matrices are discussed. Primary focus is given to novel extraction methods for some biologically active compounds including cyclic polyols, flavonoids, and oligosaccharides from plants. In addition, application of silica-, carbon-, and polymer-based solid-phase extraction adsorbents and membrane extraction for selective separation of these compounds is discussed. Potential separation process interactions are recommended; their understanding is of utmost importance for the creation of optimal conditions to extract biologically active compounds including those with estrogenic properties.

Adsorption of pharmaceuticals onto isolated polyamide active layer of NF/RO membranes.

PubMed

Liu, Yan-Ling; Wang, Xiao-Mao; Yang, Hong-Wei; Xie, Yuefeng F

2018-06-01

Adsorption of trace organic compounds (TrOCs) onto the membrane materials has a great impact on their rejection by nanofiltration (NF) and reverse osmosis (RO) membranes. This study aimed to investigate the difference in adsorption of various pharmaceuticals (PhACs) onto different NF/RO membranes and to demonstrate the necessity of isolating the polyamide (PA) active layer from the polysulfone (PS) support layer for adsorption characterization and quantification. Both the isolated PA layers and the PA+PS layers of NF90 and ESPA1 membranes were used to conduct static adsorption tests. Results showed that apparent differences existed between the PA layer and the PA+PS layer in the adsorption capacity of PhACs as well as the time necessary to reach the adsorption equilibrium. PhACs with different physicochemical properties could be adsorbed to different extents by the isolated PA layer, which was mainly attributed to electrostatic attraction/repulsion and hydrophobic interactions. The PA layer of ESPA1 exhibited apparently higher adsorption capacities for the positively charged PhACs and similar adsorption capacities for the neutral PhACs although it had significantly less total interfacial area (per unit membrane surface area) for adsorption compared to the PA layer of NF90. The higher affinity of the PA layer of ESPA1 for the PhACs could be due to its higher capacity of forming hydrogen bonds with PhACs resulted from the modified chemistry with more -OH groups. This study provides a novel approach to determining the TrOC adsorption onto the active layer of membranes for the ease of investigating adsorption mechanisms. Copyright © 2018 Elsevier Ltd. All rights reserved.

Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut

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

Schwieterman, W.; Sorrentino, D.; Potter, B.J.

1988-01-01

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (V/sub O/) of (/sup 3/H)-oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound (/sup 3/H)oleate in the medium. V/sub O/ reached a maximum as the concentration of unbound oleate was increased and was significantly inhibited both by phloretin and by prior incubation ofmore » the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 ..mu..g/ml V/sub max/ was reduced from 2480 /plus minus/ 160 to 1870 /plus minus/ 80 pmol/min per 5 /times/ 10/sup 4/ adipocytes, with no change in K/sub m/. A basic kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.« less

Unraveling Cell Processes: Interference Imaging Interwoven with Data Analysis

PubMed Central

Brazhe, A. R.; Pavlov, A. N.; Erokhova, L. A.; Yusipovich, A. I.; Maksimov, G. V.; Mosekilde, E.; Sosnovtseva, O. V.

2006-01-01

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1–0.6 Hz) result from plasma membrane processes and that higher frequency variations (20–26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties. PMID:19669463

Current Progress in Tonoplast Proteomics Reveals Insights into the Function of the Large Central Vacuole

PubMed Central

Trentmann, Oliver; Haferkamp, Ilka

2013-01-01

Vacuoles of plants fulfill various biologically important functions, like turgor generation and maintenance, detoxification, solute sequestration, or protein storage. Different types of plant vacuoles (lytic versus protein storage) are characterized by different functional properties apparently caused by a different composition/abundance and regulation of transport proteins in the surrounding membrane, the tonoplast. Proteome analyses allow the identification of vacuolar proteins and provide an informative basis for assigning observed transport processes to specific carriers or channels. This review summarizes techniques required for vacuolar proteome analyses, like e.g., isolation of the large central vacuole or tonoplast membrane purification. Moreover, an overview about diverse published vacuolar proteome studies is provided. It becomes evident that qualitative proteomes from different plant species represent just the tip of the iceberg. During the past few years, mass spectrometry achieved immense improvement concerning its accuracy, sensitivity, and application. As a consequence, modern tonoplast proteome approaches are suited for detecting alterations in membrane protein abundance in response to changing environmental/physiological conditions and help to clarify the regulation of tonoplast transport processes. PMID:23459586

Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles.

PubMed

Moche, Martin; Stremlau, Stefanie; Hecht, Lars; Göbel, Cornelia; Feussner, Ivo; Stöhr, Christine

2010-01-01

Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10-15 mM nitrate.

Avidin/PSS membrane microcapsules with biotin-binding activity.

PubMed

Endo, Yoshihiro; Sato, Katsuhiko; Sugimoto, Kentaro; Anzai, Jun-ichi

2011-08-15

Polyelectrolyte microcapsules with avidin-poly(styrene sulfonate) (PSS) membrane were prepared by a layer-by-layer deposition technique. The uptake and release of biotin-labeled fluorescein (b-FITC) as well as immobilization of biotin-labeled glucose oxidase (b-GOx) to the microcapsule were studied. The polyelectrolyte microcapsules were prepared by coating the surface of calcium carbonate (CaCO(3)) microparticles with an avidin/PSS multilayer membrane, followed by dissolution of CaCO(3) core in an ethylenediaminetetraacetic acid solution. Inner and outer poly(allylamine)/PSS films were required to isolate the microcapsules, whereas microcapsules could not be formed without the support. The uptake of b-FITC into the microcapsule was highly enhanced through a strong binding of b-FITC to avidin as compared with the uptake of biotin-free FITC. Release of b-FITC from the microcapsule was accelerated upon addition of biotin due to a competitive binding of the added biotin to the binding site of avidin. Similarly, the surface of microcapsule was modified with b-GOx with retaining its catalytic activity. Copyright © 2011 Elsevier Inc. All rights reserved.

ISOLATION OF SKELETAL MUSCLE NUCLEI

PubMed Central

Edelman, Jean C.; Edelman, P. Michael; Knigge, Karl M.; Schwartz, Irving L.

1965-01-01

A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, relatively large pestle-to-glass clearance, (b) filtering through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and (c) centrifuging in a 2.15 M sucrose-salt solution containing 0.7 mM ATP. Electron and phase-contrast microscopic observations show that the nuclei are intact, unencumbered by cytoplasmic tags, and possess well preserved distinct nucleoli, nucleoplasm, and nuclear membranes. Cytoplasmic contamination is minimal and mainly mitochondrial. Chemical assays of the nuclear fraction show that the DNA/protein and RNA/DNA ratios are comparable to those obtained in other tissues. These ratios, as well as the low specific activity obtained for cytochrome c oxidase and the virtual absence of myofibrillar ATPase, indicate a high degree of purity with minimal mitochondrial and myofibrillar contamination. The steps comprising the technique and the reasons for their selection are discussed. PMID:4287141

System and Method for Isolation of Samples

NASA Technical Reports Server (NTRS)

Zhang, Ye (Inventor); Wu, Honglu (Inventor)

2014-01-01

Systems and methods for isolating samples are provided. The system comprises a first membrane and a second membrane disposed within an enclosure. First and second reservoirs can also be disposed within the enclosure and adapted to contain one or more reagents therein. A first valve can be disposed within the enclosure and in fluid communication with the first reservoir, the second reservoir, or both. The first valve can also be in fluid communication with the first or second membranes or both. The first valve can be adapted to selectively regulate the flow of the reagents from the first reservoir, through at least one of the first and second membranes, and into the second reservoir.

Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus.

PubMed

Monte, Leonardo Garcia; Ridieri, Karine Forster; Jorge, Sérgio; Oliveira, Natasha Rodrigues; Hartwig, Daiane Drawanz; Amaral, Marta Gonçalves; Hartleben, Cláudia Pinho; Dellagostin, Odir Antonio

2015-06-01

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-12-08

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

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

PubMed Central

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

2015-01-01

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

[Isoflavone genistein-8-c-glycoside prevents the oxidative damages in structure and function of rat liver microsomal membranes].

PubMed

Zavodnik, L B

2003-01-01

Bioflavonoids (polyhydroxyphenols) are ubiquitous components of plants, fruits and vegetables; these compounds are efficient scavengers of free oxygen radicals and peroxides. The aim of this study was to investigate the antioxidant and radioprotective effects of genistein-8-C-glicoside (G8CG), an isoflavone, isolated from the flowers of Lipinus luteusl L. G8CG prevents dose-dependently the destruction of the cytochrome P-450 and its conversion to an inactive form cytochrome P-420, inhibits membrane lipid peroxidation and membrane SH-group oxidation in isolated rat liver microsomal membranes under tert-butylhydroperoxide-induced oxidative stress. Single whole-body gamma-irradiation (1 Gy) of rats results in blood plasma and liver microsomal membrane lipid peroxidation, impairments of microsomal membrane structure and function. Rat treatment with G8CG (75 mg/kg) developed the clear protective effect, stabilized membrane structure and improved the parameters of the monooxygenase function. We can conclude that G8CG can be used as antioxidant and radioprotective agent.

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase.

PubMed

Steiner, B; Lüscher, E F

1985-09-10

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.

Detergent-Based Isolation of Yeast Membrane Rafts: An Inquiry-Based Laboratory Series for the Undergraduate Cell Biology or Biochemistry Lab

ERIC Educational Resources Information Center

Willhite, D. Grant; Wright, Stephen E.

2009-01-01

Lipid rafts have been implicated in numerous cellular processes including cell signaling, endocytosis, and even viral infection. Isolation of these lipid rafts often involves detergent treatment of the membrane to dissolve nonraft components followed by separation of raft regions in a density gradient. We present here an inquiry-based lab series…

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

PubMed

Páli, Tibor; Kóta, Zoltán

2013-01-01

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

The photochemical cycle of bacteriorhodopsin

NASA Technical Reports Server (NTRS)

Lozier, R. H.; Niederberger, W.

1977-01-01

The reaction cycle of bacteriorhodopsin in the purple membrane isolated from Halobacterium halobium has been studied by optical absorption spectroscopy using low-temperature and flash kinetic techniques. After absorption of light, bacteriorhodopsin passes through at least five distinct intermediates. The temperature and pH dependence of the absorbance changes suggests that branch points and/or reversible steps exist in this cycle. Flash spectroscopy in the presence of a pH-indicating dye shows that the transient release of a proton accompanies the photoreaction cycle. The proton release occurs from the exterior and the uptake is on the cytoplasmic side of the membrane, as required by the function of bacteriorhodopsin as a light-driven proton pump. Proton translocating steps connecting release and uptake are indicated by deuterium isotope effects on the kinetics of the cycle. The rapid decay of a light-induced linear dichroism shows that a chromophore orientation change occurs during the reaction cycle.

Studies on gonadotropin receptor of rat ovary and testis

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

Zhang, Q.

1989-01-01

The subunit structure of the testicular LH/hCG receptor was studied by a chemical cross-linking technique. Leydig cells isolated from rat testis were incubated with {sup 125}I-hCG, following which the bound {sup 125}I-hCG was covalently cross-linked to the receptor on the cell surface with a cleavable or a non-cleavable cross-linking reagent. The hormone-receptor complex was extracted and then either subjected to gel permeation chromatography under nondenaturing conditions, or resolved by SDS-polyacrylamide gel electrophoresis, followed by autoradiographic analysis. The ovarian LH/hCG receptor was studied with luteal cells from pseudopregnant rats. Purification of the receptor was achieved by ligand affinity chromatography following detergentmore » solubilization of the plasma membrane. The purified hCG receptor displayed properties identical to the membrane bound receptor with regard to binding specificity and affinity, and exhibited a molecular weight of approximately 130,000 dalton.« less

Extra-corporeal membrane oxygenation-associated infections: implication of extra-intestinal pathogenic Escherichia coli clones.

PubMed

Messika, Jonathan; Clermont, Olivier; Landraud, Luce; Schmidt, Matthieu; Aubry, Alexandra; Sougakoff, Wladimir; Fernandes, Romain; Combes, Alain; Denamur, Erick; Ricard, Jean-Damien

2017-08-01

Extra-corporeal membrane oxygenation (ECMO) is a promising life-saving technique for critically ill patients. Bacterial infection is a frequent complication, and Escherichia coli the predominant causative pathogen, but little is known about the characteristics of E. coli strains in these infections. We therefore conducted a retrospective study of 33 E. coli strains responsible for 33 ECMO-related infections, in 30 subjects. Antimicrobial susceptibility, phylotyping, O-typing, clonal relatedness determination and the screening for four virulence factor genes were conducted. Polymicrobial infections were evidenced in 61.6 % of episodes, irrespective of E. coli characteristics. Extra-intestinal pathogenic strains represented the large majority (69.7 %) of all E. coli isolates. Their advantageous genetic background may explain their predominance in this context. The potential for targeted digestive decontamination should be investigated in these patients for whom infectious complications are a heavy burden.

Evidence for conductive Cl- pathways across the cell membranes of the thin ascending limb of Henle's loop.

PubMed Central

Yoshitomi, K; Kondo, Y; Imai, M

1988-01-01

To examine whether Cl- is transported via transcellular pathways in the thin ascending limb of Henle's loop (TAL), conventional microelectrode technique was applied in isolated TAL segments of hamsters perfused in vitro. The average basolateral membrane voltage (VB) was -24.5 +/- 1.5 mV (n = 18). Ouabain (10(-4) M) had no effect on VB. Sudden reduction of basolateral Cl- concentration from 165 to 5 mmol/liter caused a large depolarizing spike (+49.1 +/- 2.7 mV, n = 18), while the transepithelial potential (VT) showed lumen positive deflection by 33.4 +/- 1.2 mV, which indicates that a large Cl- conductance exists in the basolateral membrane. Reduction of luminal Cl- concentration caused sustained depolarization of luminal cell membrane from +24.5 +/- 2.1 to -9.7 +/- 3.4 mV (n = 6), which indicates that there is also a Cl- conductance in the luminal membrane. Since we have previously shown that acidification of ambient solution suppresses the transmural Cl- permeability, we tested whether acid pH also inhibits the Cl- conductance of the basolateral membrane. When pH of the bathing fluid was lowered to 5.8, the depolarizing spike of VB and the change of VT upon sudden reduction of basolateral Cl- were almost completely abolished. From these results we conclude: (a) both the luminal and the basolateral membrane of hamster TAL segments have Cl- conductances, and (b) Cl- transport in the TAL takes place, at least in part, via a transcellular route when a transepithelial Cl- gradient is present. PMID:2458388

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.

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions. Copyright © 2013 S. Karger AG, Basel.

Interactions between heavy metals and photosynthetic materials studied by optical techniques.

PubMed

Ventrella, Andrea; Catucci, Lucia; Piletska, Elena; Piletsky, Sergey; Agostiano, Angela

2009-11-01

In this work studies on rapid inhibitory interactions between heavy metals and photosynthetic materials at different organization levels were carried out by optical assay techniques, investigating the possibility of applications in the heavy metal detection field. Spinach chloroplasts, thylakoids and Photosystem II proteins were employed as biotools in combination with colorimetric assays based on dichlorophenol indophenole (DCIP) photoreduction and on fluorescence emission techniques. It was found that copper and mercury demonstrated a strong and rapid photosynthetic activity inhibition, that varied from proteins to membranes, while other metals like nickel, cobalt and manganese produced only slight inhibition effects on all tested photosynthetic materials. By emission measurements, only copper was found to rapidly influence the photosynthetic material signals. These findings give interesting information about the rapid effects of heavy metals on isolated photosynthetic samples, and are in addition to the literature data concerning the effects of growth in heavy metal enriched media.

Addressing of LnCaP Cell Using Magnetic Particles Assisted Impedimetric Microelectrode.

PubMed

Nguyen, Dung Thi Xuan; Tran, Trong Binh; Nguyen, Phuong-Diem; Min, Junhong

2016-03-01

In this study, we provide a facile, effective technique for a simple isolation and enrichment of low metastatic prostate tumor cell LNCaP using biocompatible, magnetic particles asissted impedimetric sensing system. Hydrophobic cell membrane anchors (BAM) were generated onto magnetic particles which diameters vary from 50 nm to 5 μm and were used to capture LNCaP cells from the suspension. Finally, magnetic particle-LNCaP complex were addressed onto the surface of the interdigitated microelectrode (IDM). Cell viability was monitored by our laboratory developed-technique Electrical Cell Substrate Impedance Sensing (ECIS). The results reavealed that 50 nm-magnetic particles showed best performance in terms of cell separation and cell viability. This technique provides a simple and efficient method for the direct addressing of LNCaP cell on the surface and enhances better understanding of cell behavior for cancer management in the near future.

Functional characterization of contractile vacuole isolated from Amoeba proteus.

PubMed

Nishihara, Eri; Shimmen, Teruo; Sonobe, Seiji

2004-12-01

Contractile vacuoles (CVs) released from cells of Amoeba proteus were used to analyze its function in vitro. When CV was transferred to a hypertonic medium, its volume decreased within 10 sec. When it was subsequently returned to its original medium, it quickly started swelling. However, it ruptured before recovering its initial volume. These results suggested that the CV membrane is semi-permeable and that the fluid is collected by the osmotic gradient in vivo. The water permeability of membrane of isolated CV was calculated from the rate of osmotic volume change to be 0.94 microm/sec . OsM. This high value suggested that CV membrane is equipped with water channel. CV contracted (or burst) quickly upon addition of 1 mM ATP. Contraction was induced by ATP, but not by other nucleotides, GTP, ITP, ADP, or the analogues of ATP, AMP-PNP and ATPgammaS. It was suggested that the contraction of isolated CV was caused by increase in the tension of its membrane by ATP.

Phonon Mechanisms for Excess Heat Capacity in Membrane Isolated Superconducting Transition Edge Sensors

NASA Technical Reports Server (NTRS)

Chervenak, James A.

2012-01-01

The mechanics of phonon transport in membrane-isolated superconducting transition edge sensors is discussed. Surveys of the literature on this type of sensor reveal a number of designs with excess heat capacity and a smaller subset that exhibit decoupling of the superconducting film from the underlying dielectric. A simple model is addressed in which the membrane, despite its thermal isolation, fails to fully thermalize to the temperature of the metal film heating it. A population of phonons exists which is emitted by the metal film, partially thermalizes the dielectric and is then reabsorbed in the metal film without escaping from the device structure to the thermal bath. The size of this population and its contribution to the heat capacity are estimated for several device scenarios.

Isolation of Plasma Membrane Vesicles from Mouse Placenta at Term and Measurement of System A and System β Amino Acid Transporter Activity

PubMed Central

Kusinski, L.C.; Jones, C.J.P.; Baker, P.N.; Sibley, C.P.; Glazier, J.D.

2010-01-01

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3 ± 0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function. PMID:19954844

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

A patch clamp study on reconstituted calcium permeable channels of human sperm plasma membranes.

PubMed

Ma, X H; Shi, Y L

1999-10-01

Ionic flux is thought to be important in the initiating process of gamete interaction such as acrosome reaction. However, modern electrophysiological methods, intracellular recording and patch-clamping, are difficult to approach the ion channels in mammal sperm membrane of an intact sperm due to its small size. In this work, by reconstituting the channel protein into lipid bilayer, Ca2+ channels in human spermatozoa were investigated with voltage clamp technique. Membrane proteins isolated from human sperm of 12 healthy donors were incorporated into lipid bilayer via fusion. In a cis 50//trans 10 mmol/L CaCl2 solution system, two types of channel events with similar reversal potential near the value of a perfect Ca2+ electrode, and sensitive to nifedipine and verapamil, were observed. Their unit conductance was 40 and 25 pS respectively. Percentage of channel open time was not dependent to holding potential for the former. However, for the channels of 25 pS, the percentage increased when the holding potential was changed from -20 to 100 mV. Ca(2+)-permeable channels were also detected from the spermatozoon samples of two infertile donors. Abnormal open time of these channels indicates that there are some defects in the conformation of the channel protein of infertile sperm membrane.

A simple and specific procedure to permeabilize the plasma membrane of Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Hondo, Kana; Inada, Hiroko; Chahomchuen, Thippayarat; Sekito, Takayuki; Iwaki, Tomoko; Kakinuma, Yoshimi

2009-09-01

Cu(2+)-treatment is a useful technique in selectively permeabilizing the fungal plasma membrane. We describe herein a practical application with Schizosaccharomyces pombe. Incubation of cells with 0.5 mM CuCl(2) at 30 degrees C for 20 min induced efficient leakage of cytosolic constituents. The kinetic characteristics of the calcium and amino acid flux from Cu(2+)-treated S. pombe cells suggested that the Cu(2+) treatment permeabilized the plasma membrane without loss of vacuolar function. As a further application of the method, the amino acid contents of Cu(2+)-treated and untreated cells were also determined. The amino acid pool of Cu(2+)-treated wild-type cells was enriched in basic amino acids but not in acidic amino acids, as is characteristic of the vacuolar amino acid pool of fungi, including Saccharomyces cerevisiae and Neurosporra crassa. The amino acid pool of the S. pombe V-ATPase mutant vma1Delta was also successfully determined. We conclude that the vacuolar amino acid pool of S. pombe can be measured using Cu(2+)-treated cells. The method is simple, inexpensive, and rapid relative to the isolation of vacuolar vesicles, making it useful in estimating vacuolar pools and transport across the vacuolar membrane.

Microfabrication of a High-Throughput Nanochannel Delivery/Filtration System

NASA Technical Reports Server (NTRS)

Ferrari, Mauro; Liu, Xuewu; Grattoni, Alessandro; Fine, Daniel; Hosali, Sharath; Goodall, Randi; Medema, Ryan; Hudson, Lee

2011-01-01

A microfabrication process is proposed to produce a nanopore membrane for continuous passive drug release to maintain constant drug concentrations in the patient s blood throughout the delivery period. Based on silicon microfabrication technology, the dimensions of the nanochannel area, as well as microchannel area, can be precisely controlled, thus providing a steady, constant drug release rate within an extended time period. The multilayered nanochannel structures extend the limit of release rate range of a single-layer nanochannel system, and allow a wide range of pre-defined porosity to achieve any arbitrary drug release rate using any preferred nanochannel size. This membrane system could also be applied to molecular filtration or isolation. In this case, the nanochannel length can be reduced to the nanofabrication limit, i.e., 10s of nm. The nanochannel delivery system membrane is composed of a sandwich of a thin top layer, the horizontal nanochannels, and a thicker bottom wafer. The thin top layer houses an array of microchannels that offers the inlet port for diffusing molecules. It also works as a lid for the nanochannels by providing the channels a top surface. The nanochannels are fabricated by a sacrificial layer technique that obtains smooth surfaces and precisely controlled dimensions. The structure of this nanopore membrane is optimized to yield high mechanical strength and high throughput.

High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

PubMed Central

2012-01-01

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

Desipramine prevents cardiac gap junction uncoupling.

PubMed

Jozwiak, Joanna; Dietze, Anna; Grover, Rajiv; Savtschenko, Alex; Etz, Christian; Mohr, Friedrich W; Dhein, Stefan

2012-11-01

Uncoupling of cardiac gap junction channels is an important arrhythmogenic mechanism in ischemia/reperfusion. Antiarrhythmic peptide AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)) has been shown to prevent acidosis-induced uncoupling and ischemia-related increase in dispersion. Previous structure-effect investigations and subsequent computer modeling studies indicated that the tricyclic antidepressant desipramine may exert similar effects as AAP10. We assessed the binding of (14)C-AAP10 to membranes of rabbit cardiac ventricles and its displacement with desipramine in a classical radioligand binding and competition study. Gap junction currents were measured between isolated pairs of human atrial cardiomyocytes under normal and acidotic (pH 6.3) conditions with or without 1 μmol/l desipramine using dual whole-cell voltage clamp. The effect of 1 μmol/l desipramine was assessed in isolated rabbit hearts (Langendorff technique) undergoing local ischemia by coronary occlusion with 256-channel electrophysiological mapping and subsequent analysis of connexin43 (Cx43) expression, phosphorylation (Western blot), and subcellular localization (immunohistology). We found saturable (14)C-AAP10 binding to cardiac membranes (K (D), 0.29 ± 0.11 nmol/l; B (max), 42.5 ± 7.2 pmol/mg) which could be displaced by desipramine with a K (D.High) = 0.14 μmol/l and a K (D.Low) = 22 μmol/l. Acidosis reduced the gap junction conductance in human cardiomyocyte pairs from 24.1 ± 4.7 to 11.5 ± 2.5 nS, which could be significantly reversed by desipramine (26.6 ± 4.8 nS). In isolated hearts, ischemia resulted in significantly increased dispersion of activation-recovery intervals, loss of membrane Cx43, and dephosphorylation of Cx43, which all could be prevented by desipramine. Desipramine seems to prevent the uncoupling of cardiac gap junctions and ischemia-related increase in dispersion.

Antimicrobial peptide from mucus of Andrias davidianus: screening and purification by magnetic cell membrane separation technique.

PubMed

Pei, Jinjin; Jiang, Lei

2017-07-01

Andrias davidianus, the Chinese giant salamander, has been used in traditional Chinese medicine for many decades. However, no antimicrobial peptides (AMPs) have been described from A. davidianus until now. Here we describe a novel AMP (andricin 01) isolated from the mucus of A. davidianus. The peptide was recovered using an innovative magnetic cell membrane separation technique and was characterised using mass spectrometry and circular dichroism (CD) spectroscopy. Andricin 01 is comprised of ten amino acid residues with a total molecular mass of 955.1 Da. CD spectrum analysis gave results similar to the archetypal random coil spectrum, consistent with the three-dimensional rendering calculated by current bioinformatics tools. Andricin 01 was found to be inhibitory both to Gram-negative and Gram-positive bacteria. Furthermore, the peptide at the minimal bacterial concentration did not show cell cytotoxicity against human hepatocytes or renal cells and did not show haemolytic activity against red blood cells, indicating that is potentially safe and effective for human use. Andricin 01 shows promise as a novel antibacterial that may provide an insight into the development of new drugs. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Force-controlled patch clamp of beating cardiac cells.

PubMed

Ossola, Dario; Amarouch, Mohamed-Yassine; Behr, Pascal; Vörös, János; Abriel, Hugues; Zambelli, Tomaso

2015-03-11

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Magnetic Tissue Engineering for Voice Rehabilitation - First Steps in a Promising Field.

PubMed

Dürr, Stephan; Bohr, Christopher; Pöttler, Marina; Lyer, Stefan; Friedrich, Ralf Philipp; Tietze, Rainer; Döllinger, Michael; Alexiou, Christoph; Janko, Christina

2016-06-01

The voice is one of the most important instruments of communication between humans. It is the product of intact and well-working vocal folds. A defect of these structures causes dysphonia, associated with a clear reduction of quality of life. Tissue engineering of the vocal folds utilizing magnetic cell levitation after nanoparticle loading might be a technique to overcome this challenging problem. Vocal fold fibroblasts (VFFs) were isolated from rabbit larynges and cultured. For magnetization, cells were incubated with superparamagnetic iron oxide nanoparticles (SPION) and the loading efficiency was determined by Prussian blue staining. Biocompatibility was analyzed in flow cytometry by staining with annexin V-fluorescein isothiocyanate propidium iodide, 1,1',3,3,3',3'-hexamethylindodicarbo-cyanine iodide [DiIC1(5)] and propidium idodide-Triton X-100 to monitor phosphatidylserine exposure, plasma membrane integrity, mitochondrial membrane potential and DNA degradation. Isolated VFFs can be successfully loaded with SPION, and optimal iron loading associated with minimized cytotoxicity represents a balancing act in magnetic tissue engineering. Our data are a firm basis for the next steps of investigations. Magnetic tissue engineering using magnetic nanoparticle-loaded cells which form three-dimensional structures in a magnetic field will be a promising approach in the future. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

PubMed

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure.

PubMed

Pavez, Monica; Vieira, Camila; de Araujo, Maria Rita; Cerda, Alvaro; de Almeida, Lara Mendes; Lincopan, Nilton; Mamizuka, Elsa Masae

2016-07-01

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates.

PubMed

Lavigne, J-P; Sotto, A; Nicolas-Chanoine, M-H; Bouziges, N; Bourg, G; Davin-Regli, A; Pagès, J-M

2012-06-01

Imipenem-susceptible E. aerogenes isolates exhibiting extended spectrum β-lactamases, target mutations and a basal efflux expression, were identified in five patients. After imipenem treatment, imipenem-intermediate susceptible (IMI-I) or resistant (IMI-R) isolates emerged in these patients. Alteration in porin synthesis and increase in efflux expression were observed in the IMI-I isolates whereas complete loss of the porins, LPS alteration and efflux overexpression were observed in the IMI-R isolates. Bacterial virulence of the strains was investigated by the Caenorhabditis elegans model. The IMI-R isolates were shown to be significantly less virulent than the IMI-susceptible or IMI-I isolates. The pleiotropic membrane alteration and its associated fitness burden exhibited by E. aerogenes isolates influence their antibiotic resistance and their virulence behaviour. These findings highlight the balance between the low permeability-related resistance and virulence and their relationships with the treatment of resistant pathogens. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Structure of the Human Mitochondrial Ribosome Studied In Situ by Cryoelectron Tomography.

PubMed

Englmeier, Robert; Pfeffer, Stefan; Förster, Friedrich

2017-10-03

Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated mammalian mitoribosome, but its mode of membrane association remained hypothetical. In this study, we used cryoelectron tomography to visualize human mitoribosomes in isolated mitochondria. The subtomogram average of the membrane-associated human mitoribosome reveals a single major contact site with the inner membrane, mediated by the mitochondria-specific protein mL45. A second rRNA-mediated contact site that is present in yeast is absent in humans, resulting in a more variable association of the human mitoribosome with the inner membrane. Despite extensive structural differences of mammalian and fungal mitoribosomal structure, the principal organization of peptide exit tunnel and the mL45 homolog remains invariant, presumably to align the mitoribosome with the membrane-embedded insertion machinery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Physicochemical properties and membrane biofouling of extra-cellular polysaccharide produced by a Micrococcus luteus strain.

PubMed

Feng, Lei; Li, Xiufen; Song, Ping; Du, Guocheng; Chen, Jian

2014-07-01

The physicochemical properties of the extra-cellular polysaccharide (EPS) produced by a Micrococcus luteus strain, a dominating strain isolated from membrane biofouling layer, were determined in this study. The EPS isolated from this strain was measured to have an average molecular weight of 63,540 Da and some typical polysaccharide absorption peaks in Fourier transform infrared spectrum. Monosaccharide components of the EPS contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. Pseudo plastic properties were also observed for the EPS through the rheological measurement. The EPS was further characterized for its behavior to cause membrane flux decline. The results showed that both flux declines for polyvinylidenefluoride (PVDF) and polypropylene membranes became more severe as EPS feed concentration increased. A higher irreversible fouling for the PVDF membrane suggested that the EPS had the larger fouling potential to this microfiltration membrane.

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells.

PubMed

Morré, D M; Morre, D J

2000-06-23

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

NASA Technical Reports Server (NTRS)

Morre, D. M.; Morre, D. J.

2000-01-01

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum

Isolation of syncytial microvillous membrane vesicles from human term placenta and their application in drug-nutrient interaction studies.

PubMed

van der Aa, E M; Copius Peereboom-Stegeman, J H; Russel, F G

1995-09-01

The initial step in placental uptake of nutrients occurs across the syncytial microvillous membrane of the trophoblast. This study was designed to isolate syncytial microvillous membrane vesicles (SMMV) of human term placenta, to validate their purity and viability, and to investigate the interaction of several commonly used drugs with the transport of two essential nutrients: alanine and choline. SMMV were isolated according to an established procedure, but instead of homogenization the initial preparation step was replaced by mincing of placental tissue followed by gently stirring to loosen the microvilli. These modifications doubled the protein recovery and increased the enrichment in alkaline phosphatase, whereas no substantial contamination with basal membranes nor interfering subcellular organelles was found. The functional viability of the vesicles was evaluated through the transport of alanine. In accordance with literature, uptake was sodium-dependent, inhibitable by structural analogues, and saturable. A number of cationic drugs were were able to able to inhibit choline uptake, whereas no effect on alanine transport was observed. Anionic drugs, drugs of abuse, and catecholamines did not interfere with alanine transport either. In conclusion, our isolated SMMV provide a suitable tool for screening drug-nutrient interactions at the level of membrane transport. In view of the very low susceptibility of the alanine transporter to drug inhibition and the relatively high drug concentrations necessary to inhibit choline transport, it seems unlikely that clinically important drug interactions may occur with these nutrients.

Influence of beta-radiation sterilisation in properties of new chitosan/soybean protein isolate membranes for guided bone regeneration.

PubMed

Silva, R M; Elvira, C; Mano, J F; San Román, J; Reis, R L

2004-04-01

Novel chitosan (cts) and soybean protein isolate (SI) blended membranes were prepared. These membranes were produced by solvent casting. Besides combining the advantages of both materials, cts/SI membranes exhibit a biphasic structure that will eventually originate in situ porous formation, through a two-step degradation mechanism. In this particular work the effect of beta-radiation over the properties of these membranes was evaluated. beta-radiation sterilisation was performed at three different doses (25, 50 and 100 kGy) and eventual surface chemical changes were evaluated by Fourier transformed infrared--with attenuated total reflection and contact angle measurements. Moreover, eventual bulk properties changes due to beta-radiation were assessed by means of mechanical tensile tests and water uptake measurements. In general, no substantial changes were detected on the studied properties, with the exception of the surface energy that was found to be slightly increased for higher applied doses.

Potassium accumulation by the glial membrane pump as revealed by membrane potential recording from isolated rabbit retinal Müller cells.

PubMed

Reichenbach, A; Nilius, B; Eberhardt, W

1986-01-30

Müller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. In a special perfusion chamber, the cells were penetrated with a recording electrode. When high-K+ solutions were applied into the environment of the cells by means of a second micropipette, the cell membrane depolarized strongly. During prolonged application of high-K+ solutions, however, there occurred a marked repolarization, and after cessation of high-K+ application, a strong hyperpolarization was observed. Both effects disappeared under the influence of ouabain, suggesting the accumulation of intracellular K+ by an active membrane pump. The data were used for calculation of the membrane's Na+:K+ permeability ratio, the intracellular K+ concentration, the pump rate and the mean pump site density. The calculated values are in good agreement with published data from mammalian astrocytes and are compared with those from amphibian Müller cells.

Characterization of brush borders purified in iso-osmotic medium and microvillar membranes subfractionated from mouse small intestine.

PubMed Central

Fujita, M; Ohta, H; Uezato, T

1981-01-01

Brush borders free of nuclei were isolated by repeated homogenization and centrifugation in iso-osmotic medium. They showed typical morphology under electron microscopy. The mean recovery and enrichment of alkaline phosphatase activity in the brush-border fraction were 50% and 17.5-fold respectively. gamma-Glutamyl transpeptidase showed a close parallelism with alkaline phosphatase and sucrase in subcellular distribution. Microvillar membranes were purified from isolated brush borders; they showed a further enrichment for alkaline phosphatase and were composed of homogeneous vesicles. Both brush-border and microvillar-membrane preparations were analysed for contamination by basolateral and endoplasmic-reticular membranes. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the microvillar-membrane preparation in six different systems revealed approx. 40 components in the mol.wt. range 15 000-232 000. They were grouped into seven major classes on the basis of molecular weight and electrophoretic patterns. Images PLATE 1 PLATE 2 PMID:7317008

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

PubMed Central

Kim, Hyung-Sik; Kang, Kyung-Sun

2012-01-01

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine. PMID:23024756

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

PubMed

Jonas, E A; Knox, R J; Kaczmarek, L K

1997-07-01

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

PubMed

Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

2014-03-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

PubMed Central

Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

2014-01-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines.

PubMed

Grancara, Silvia; Ohkubo, Shinji; Artico, Marco; Ciccariello, Mauro; Manente, Sabrina; Bragadin, Marcantonio; Toninello, Antonio; Agostinelli, Enzo

2016-10-01

Mitochondria represent cell "powerhouses," being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca(2+) transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism. Mitochondrial permeability transition drives both apoptosis and necrosis, whereas mitochondrial outer membrane permeability is characteristic of apoptosis. Adenine nucleotide translocase remains the most important component involved in membrane permeability, with the opening of the transition pore, although other proteins, such as ATP synthase or phosphate carriers, have been proposed. Intrinsic cell death is triggered by the release from mitochondria of proteic factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO, with the activation of caspases upon mitochondrial permeability transition or mitochondrial outer membrane permeability induction. Mitochondrial permeability transition induces the permeability of the inner membrane in sites in contact with the outer membrane; mitochondrial outer membrane permeability forms channels on the outer membrane by means of various stimuli involving Bcl-2 family proteins. The biologically active amines, spermine, and agmatine, have specific functions on mitochondria which distinguish them from other amines. Enzymatic oxidative deamination of spermine by amine oxidases in tumor cells may produce reactive oxygen species, leading to transition pore opening and apoptosis. This process could be exploited as a new therapeutic strategy to combat cancer.

Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure.

PubMed

Vildhede, Anna; Wiśniewski, Jacek R; Norén, Agneta; Karlgren, Maria; Artursson, Per

2015-08-07

Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.

Differential Expression and Immunolocalization of Antioxidant Enzymes in Entamoeba histolytica Isolates during Metronidazole Stress

PubMed Central

Iyer, Lakshmi Rani; Singh, Nishant; Verma, Anil Kumar; Paul, Jaishree

2014-01-01

Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress. PMID:25013795

Impact of Detergents on Membrane Protein Complex Isolation.

PubMed

Lee, Yu-Chen; Bååth, Jenny Arnling; Bastle, Ryan M; Bhattacharjee, Sonali; Cantoria, Mary Jo; Dornan, Mark; Gamero-Estevez, Enrique; Ford, Lenzie; Halova, Lenka; Kernan, Jennifer; Kürten, Charlotte; Li, Siran; Martinez, Jerahme; Sachan, Nalani; Sarr, Medoune; Shan, Xiwei; Subramanian, Nandhitha; Rivera, Keith; Pappin, Darryl; Lin, Sue-Hwa

2018-01-05

Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Furthermore, we compared the effects of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents except Brij-35 could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that β-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij-35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.

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

PubMed

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

2014-01-01

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

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile.

PubMed

Bastardo, A; Bohle, H; Ravelo, C; Toranzo, A E; Romalde, J L

2011-02-22

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.

[Preparation of chicken red blood cells for calibration of flow cytometry].

PubMed

Yin, Jian; Zhao, Shutao; Wu, Xiaodong; Wang, Ce; Wu, Yunliang

2013-01-01

To prepare stable chicken red blood cells for the calibration of flow cytometry. The traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells. Chicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01]. The chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-04-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed Central

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-01-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains. Images PMID:3980081

Negative Inotropic Effects of High Mobility Box Group 1 Protein in Isolated Contracting Cardiac Myocytes

PubMed Central

Tzeng, Huei-Ping; Fan, Jinping; Vallejo, Jesus G.; Dong, Jian Wen; Chen, Xiongwen; Houser, Steven R.; Mann, Douglas L.

2013-01-01

HMGB1 released from necrotic cells or macrophages functions as a late inflammatory mediator, and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients using fluo-3, and L-type calcium currents using whole cell perforate configuration of the patch clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca++ transient within 5 min (p <0.01). The immediate negative inotropic effects HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward L-type calcium currents also was documented by the patch clamp technique. HMGB1 induced the PKCε translocation and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx, and suggest that HMGB1 maybe act as a novel myocardial depressant factor during cardiac injury. PMID:18223193

Measuring Mitochondrial Function in Permeabilized Cells Using the Seahorse XF Analyzer or a Clark-Type Oxygen Electrode.

PubMed

Divakaruni, Ajit S; Rogers, George W; Murphy, Anne N

2014-05-27

Measurements of mitochondrial respiration in intact cells can help define metabolism and its dysregulation in fields such as cancer, metabolic disease, immunology, and neurodegeneration. Although cells can be offered various substrates in the assay medium, many cell types can oxidize stored pools of energy substrates. A general bioenergetic profile can therefore be obtained using intact cells, but the inability to control substrate provision to the mitochondria can restrict an in-depth, mechanistic understanding. Mitochondria can be isolated from intact cells, but the yield and quality of the end product is often poor and prone to subselection during isolation. Plasma membrane permeabilization of cells provides a solution to this challenge, allowing experimental control of the medium surrounding the mitochondria. This unit describes techniques to measure respiration in permeabilized adherent cells using a Seahorse XF Analyzer or permeabilized suspended cells in a Hansatech Oxygraph. Copyright © 2014 John Wiley & Sons, Inc.

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

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

Wheeler, J.J.; Boss, W.F.

1987-10-01

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-(2-/sup 3/H)inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in (/sup 3/H)inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP/sub 2/). An additional (/sup 3/H)inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP/sub 2/ on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily inmore » the lower phase, microsomal/mitchrondrial-rich fraction.« less

Significance of Fecal Coliform-Positive Klebsiella1

PubMed Central

Bagley, Susan T.; Seidler, Ramon J.

1977-01-01

A total of 191 Klebsiella pneumoniae isolates of human clinical, bovine mastitis, and a wide variety of environmental sources were tested for fecal coliform (FC) response with the membrane filtration and most probable number techniques. Twenty-seven Escherichia coli cultures of human clinical and environmental origins were also tested. Eighty-five percent (49/58) of known pathogenic K. pneumoniae were FC positive, compared with 16% (19/120) of the environmental strains. E. coli results indicated 93% (13/14) of the clinical and 85% (11/13) of the environmental strains as FC positive. There was no significant difference in the incidence of FC-positive cultures between pathogenic Klebsiella and E. coli. pH measurements of K. pneumoniae and E. coli cultures growing in m-FC broth at 44.5°C revealed three distinct pH ranges correlating with colony morphology. β-Galactosidase assays of Klebsiella and E. coli cultures at 44.5°C indicated all were able to hydrolyze lactose, even if they were FC negative by the membrane filtration or most probable number techniques. The FC response pattern appears stable in K. pneumoniae. Three pathogenic cultures showed no change in FC responses after 270 generations of growth in sterile pulp mill effluent. Since K. pneumoniae is carried in the gastrointestinal tract of humans and animals and 85% of the tested pathogenic strains were FC positive, the isolation of FC-positive Klebsiella organisms from the environment would indicate their fecal or clinical origin or both. The added fact that K. pneumoniae is an opportunistic pathogen of increasing importance makes the occurrence of FC-positive environmental Klebsiella, particularly in large numbers, a potential human and animal health hazard. PMID:18086

Fourier transform infrared spectroscopic studies of the secondary structure and thermal denaturation of CaATPase from rabbit skeletal muscle

NASA Astrophysics Data System (ADS)

Jaworsky, Mark; Brauner, Joseph W.; Mendelsohn, Richard

Fourier transform i.r. spectroscopy has been used to monitor structural alterations induced by thermal denaturation of the intrinsic membrane protein CaATPase in aqueous media. The protein has been isolated, purified and studied in five forms: (i) In its native lipid environment after isolation from rabbit sarcoplasmic reticulum, both in H 2O and D 2O suspensions. (ii) After both mild and extensive tryptic digestion has cleaved those residues external to the membrane bilayer. (iii) Reconstituted in vesicle form with bovine brain sphingomyelin. Fourier deconvolution techniques have been used to enhance the resolution of the intrinsically overlapped Amide I and Amide II spectral regions. Large spectral alterations apparent in the deconvoluted spectra occur in these regions upon thermal denaturation of the protein which are consistent with the formation of a large proportion of β-antiparallel sheet form. The alteration parallels the loss in ATPase activity. A mild tryptic digestion increases slightly the proportion of α-helix and/or random coil secondary structure. A thermal transition to a form containing a high proportion of β structure is still evident. Extensive tryptic digestion nearly abolishes the alpha helical plus random coil secondary structure, while producing a high proportion of β form which is resistant to further thermally induced structural alterations. Studies of CaATPase reconstituted into vesicles with bovine brain sphingomyelin reveal a higher proportion of β structure than the native enzyme, with further introduction of β structure on thermal denaturation. Both the utility of deconvolution techniques and the necessity for caution in their application are apparent from the current experiments.

Lipid-Coated Gold Nanoparticles and FRET Allow Sensitive Monitoring of Liposome Clustering Mediated by the Synaptotagmin-7 C2A Domain.

PubMed

Hamilton, Desmond J; Coffman, Matthew D; Knight, Jefferson D; Reed, Scott M

2017-09-12

Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca 2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Förster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein-membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca 2+ requirements: the threshold Ca 2+ concentration for membrane binding is lowest, followed by apposition and aggregation. However, aggregation is highly sensitive to protein concentration and can occur even at submicromolar Syt7 C2A; thus, highly sensitive assays are needed for measuring apposition without complications arising from aggregation. Notably, the localized surface plasmon resonance of the LCAuNP is sensitive to ≤10 nM Syt7 C2A concentrations. Furthermore, when the LCAuNPs were added into a FRET-based liposome apposition assay, the resultant energy transfer increased; possible explanations are discussed. Overall, LCAuNP-based methods allow for highly sensitive detection of protein-induced membrane apposition under conditions that miminize large-scale aggregation.

L-Carnitine suppresses oleic acid-induced membrane permeability transition of mitochondria.

PubMed

Oyanagi, Eri; Yano, Hiromi; Kato, Yasuko; Fujita, Hirofumi; Utsumi, Kozo; Sasaki, Junzo

2008-10-01

Membrane permeability transition (MPT) of mitochondria has an important role in apoptosis of various cells. The classic type of MPT is characterized by increased Ca(2+) transport, membrane depolarization, swelling, and sensitivity to cyclosporin A. In this study, we investigated whether L-carnitine suppresses oleic acid-induced MPT using isolated mitochondria from rat liver. Oleic acid-induced MPT in isolated mitochondria, inhibited endogenous respiration, caused membrane depolarization, and increased large amplitude swelling, and cytochrome c (Cyt. c) release from mitochondria. L-Carnitine was indispensable to beta-oxidation of oleic acid in the mitochondria, and this reaction required ATP and coenzyme A (CoA). In the presence of ATP and CoA, L-carnitine stimulated oleic acid oxidation and suppressed the oleic acid-induced depolarization, swelling, and Cyt. c release. L-Carnitine also contributed to maintaining mitochondrial function, which was decreased by the generation of free fatty acids with the passage of time after isolation. These results suggest that L-carnitine acts to maintain mitochondrial function and suppresses oleic acid-mediated MPT through acceleration of beta-oxidation. Copyright (c) 2008 John Wiley & Sons, Ltd.

In vitro mitochondrial test to assess haemodialyser biocompatibility.

PubMed

Tabouy, L J; Chauvet-Monges, A M; Brunet, P J; Braguer, D L; García, P A; Berland, Y F; Crevat, A D

1997-08-01

This paper describes an in vitro mitochondrial test to assess the biocompatibility of haemodialysers. We tested on isolated liver mitochondria the effect of solutions obtained by an aqueous rinse of different haemodialysers (cuprophane, cellulose acetate, Hemophan, polyacrylonitrile, polymethylmethacrylate, polysulphone, polyamide). Moreover, to determine the penetration into the cell and the cytotoxicity of these solutions from haemodialysers, we examined the effect of rinse solutions on HT29-D4 cells. Our results showed that rinse solutions from haemodialysers decrease the mitochondrial ATP synthesis. Cuprophane has the most marked effect, and the synthetic membranes exhibited only mild effects. Rinse solutions penetrated the cell and were cytotoxic by acting on mitochondria in the cell. In this respect, cellulosic membranes were the most toxic. Taken together our findings lead to a classification of haemodialyser membranes which is identical to one based on criteria such as activation of complement (cuprophane > other cellulosics > synthetics). Moreover isolated mitochondria make it possible to differentiate among the synthetic membranes. Isolated mitochondria thus appear to be a good in vitro test to assess the biocompatibility of haemodialysers.

Adaptation and Molecular Characterization of Two Malaysian Very Virulent Infectious Bursal Disease Virus Isolates Adapted in BGM-70 Cell Line

PubMed Central

Lawal, Nafi'u; Arshad, Siti Suri

2017-01-01

Two Malaysian very virulent infectious bursal disease virus (vvIBDV) strains UPM0081 and UPM190 (also known as UPMB00/81 and UPM04/190, respectively) isolated from local IBD outbreaks were serially passaged 12 times (EP12) in specific pathogen free (SPF) chicken embryonated eggs (CEE) by chorioallantoic membrane (CAM) route. The EP12 isolate was further adapted and serially propagated in BGM-70 cell line up to 20 passages (P20). Characteristic cytopathic effects (CPEs) were subtly observed at P1 in both isolates 72 hours postinoculation (pi). The CPE became prominent at P5 with cell rounding, cytoplasmic vacuoles, granulation, and detachment from flask starting from day 3 pi, up to 7 days pi with titers of 109.50 TCID50/mL and log109.80 TCID50/mL for UPM0081 and UPM190, respectively. The CPE became subtle at P17 and disappeared by P18 and P19 for UPM0081 and UPM190, respectively. However, the presence of IBDV was confirmed by immunoperoxidase, immunofluorescence, and RT-PCR techniques. Phylogenetic analysis showed that these two isolates were of the vvIBDV. It appears that a single mutation of UPM190 and UPM0081 IBDV isolates at D279N could facilitate vvIBDV strain adaptability in CEE and BGM-70 cultures. PMID:29230245

Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

PubMed

Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

2017-10-01

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy.

PubMed

Oh, Yoo Jin; Plochberger, Birgit; Rechberger, Markus; Hinterdorfer, Peter

2017-06-01

Lipopolysaccharide (LPS) on gram-negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano-resolution approaches in microbiology. Copyright © 2017 John Wiley & Sons, Ltd.

Gestational undernourishment modifies the composition of skeletal muscle transverse tubule membranes and the mechanical properties of muscles in newborn rats.

PubMed

Ramírez-Oseguera, Ricardo Tonathiu; Jiménez-Garduño, Aura Matilde; Alvarez, Rocío; Heine, Katharina; Pinzón-Estrada, Enrique; Torres-Saldaña, Ismael; Ortega, Alicia

2013-01-01

[corrected] Skeletal muscle (SM) constitutes more than 40% of the body weight in adulthood. Transports dietary glucose mainly through the insulin-dependent glucose transporter (Glut-4) located in the Transverse tubule membrane system (TT). The TT development ends shortly after birth. The TT membrane hosts the proteins involved in excitation-contraction coupling and glucose uptake. Glycaemic regulation through movement is a key function of fully developed skeletal muscle. In this study, we aimed to characterize the effect of gestational undernourishment (GUN) in rats GLUT-4 expression and on the protein/lipid content of the TT membranes. We also examined the effect of GUN on the mechanical properties of muscles as an indication of the metabolic condition of the SM at birth. Isolated TT membrane from SM of GUN rats were used to study lipid/protein content and protein stability by differential scanning calorimetry. The effect of GUN on the SM mechanical properties was determined in isolated Extensor Digitorum Longus (EDL) muscle. We demonstrate that compared to control, GUN in the new-born produces; i) decreases body weight; ii) diminution in SM mass; iii) decreases the formation of TT membranes; iv) expresses TT membrane proteins with higher thermal stability. The TT membrane expression of GLUT-4 in GUN offspring was twice that of controls. The isolated EDL of GUN offspring was 20% stronger as measured by contractile force and more resistant to fatigue relative to controls. These results provide the first evidence of adaptive changes of the SM in new-borns exposed to severe gestational food restriction. The effects of GUN on muscle at birth are the first step toward detrimental SM metabolic function, contributing to the physiopathology of metabolic diseases in adulthood. © 2013 S. Karger AG, Basel

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

PubMed

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

2018-04-29

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

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis.

PubMed

Yu, Daoyong; Huang, Guihong; Xu, Fengxi; Wang, Mengfei; Liu, Shuang; Huang, Fang

2014-06-01

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

PubMed

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

2000-04-01

Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

PubMed

Brett, Sabine I; Lucien, Fabrice; Guo, Charles; Williams, Karla C; Kim, Yohan; Durfee, Paul N; Brinker, C J; Chin, Joseph I; Yang, Jun; Leong, Hon S

2017-05-01

The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches. © 2017 Wiley Periodicals, Inc.

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

PubMed

Hardy, M E L; Lawrence, C L; Standen, N B; Rodrigo, G C

2006-01-01

Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.

Laser-induced lipolysis on adipose cells

NASA Astrophysics Data System (ADS)

Solarte, Efrain; Gutierrez, O.; Neira, Rodrigo; Arroyave, J.; Isaza, Carolina; Ramirez, Hugo; Rebolledo, Aldo F.; Criollo, Willian; Ortiz, C.

2004-10-01

Recently, a new liposuction technique, using a low-level laser (LLL) device and Ultrawet solution prior to the procedure, demonstrated the movement of fat from the inside to the outside of the adipocyte (Neira et al., 2002). To determine the mechanisms involved, we have performed Scanning and Transmission Electron Microscopy studies; Light transmittance measurements on adipocyte dilutions; and a study of laser light propagation in adipose tissue. This studies show: 1. Cellular membrane alterations. 2. LLL is capable to reach the deep adipose tissue layer, and 3. The tumescence solution enhances the light propagation by clearing the tissue. MRI studies demonstrated the appearance of fat on laser treated abdominal tissue. Besides, adipocytes were cultivated and irradiated to observe the effects on isolated cells. These last studies show: 1. 635 nm-laser alone is capable of mobilizing cholesterol from the cell membrane; this action is enhanced by the presence of adrenaline and lidocaine. 2. Intracellular fat is released from adipocytes by co joint action of adrenaline, aminophyline and 635 nm-laser. Results are consistent with a laser induced cellular process, which causes fat release from the adipocytes into the intercellular space, besides the modification of the cellular membranes.

Detecting protein-protein interactions in the intact cell of Bacillus subtilis (ATCC 6633).

PubMed

Winters, Michael S; Day, R A

2003-07-01

The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C(2)N(2)) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins.

Detecting Protein-Protein Interactions in the Intact Cell of Bacillus subtilis (ATCC 6633)

PubMed Central

Winters, Michael S.; Day, R. A.

2003-01-01

The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C2N2) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins. PMID:12837803

The spine neck filters membrane potentials.

PubMed

Araya, Roberto; Jiang, Jiang; Eisenthal, Kenneth B; Yuste, Rafael

2006-11-21

Dendritic spines receive most synaptic inputs in the forebrain. Their morphology, with a spine head isolated from the dendrite by a slender neck, indicates a potential role in isolating inputs. Indeed, biochemical compartmentalization occurs at spine heads because of the diffusional bottleneck created by the spine neck. Here we investigate whether the spine neck also isolates inputs electrically. Using two-photon uncaging of glutamate on spine heads from mouse layer-5 neocortical pyramidal cells, we find that the amplitude of uncaging potentials at the soma is inversely proportional to neck length. This effect is strong and independent of the position of the spine in the dendritic tree and size of the spine head. Moreover, spines with long necks are electrically silent at the soma, although their heads are activated by the uncaging event, as determined with calcium imaging. Finally, second harmonic measurements of membrane potential reveal an attenuation of somatic voltages into the spine head, an attenuation directly proportional to neck length. We conclude that the spine neck plays an electrical role in the transmission of membrane potentials, isolating synapses electrically.

The spine neck filters membrane potentials

PubMed Central

Araya, Roberto; Jiang, Jiang; Eisenthal, Kenneth B.; Yuste, Rafael

2006-01-01

Dendritic spines receive most synaptic inputs in the forebrain. Their morphology, with a spine head isolated from the dendrite by a slender neck, indicates a potential role in isolating inputs. Indeed, biochemical compartmentalization occurs at spine heads because of the diffusional bottleneck created by the spine neck. Here we investigate whether the spine neck also isolates inputs electrically. Using two-photon uncaging of glutamate on spine heads from mouse layer-5 neocortical pyramidal cells, we find that the amplitude of uncaging potentials at the soma is inversely proportional to neck length. This effect is strong and independent of the position of the spine in the dendritic tree and size of the spine head. Moreover, spines with long necks are electrically silent at the soma, although their heads are activated by the uncaging event, as determined with calcium imaging. Finally, second harmonic measurements of membrane potential reveal an attenuation of somatic voltages into the spine head, an attenuation directly proportional to neck length. We conclude that the spine neck plays an electrical role in the transmission of membrane potentials, isolating synapses electrically. PMID:17093040

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.

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem

PubMed Central

Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

2015-01-01

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen. PMID:26398358

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem.

PubMed

Philippe, Nadège; Maigre, Laure; Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

2015-01-01

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen.

Cleistopholine isolated from Enicosanthellum pulchrum exhibits apoptogenic properties in human ovarian cancer cells.

PubMed

Nordin, Noraziah; Majid, Nazia Abdul; Mohan, Syam; Dehghan, Firouzeh; Karimian, Hamed; Rahman, Mashitoh Abdul; Ali, Hapipah Mohd; Hashim, Najihah Mohd

2016-04-15

Cleistopholine is a natural alkaloid present in plants with numerous biological activities. However, cleistopholine has yet to be isolated using modern techniques and the mechanism by which this alkaloid induces apoptosis in cancer cells remains to be elucidated. This study aims to isolate cleistopholine from the roots of Enicosanthellum pulchrum by using preparative-HPLC technique and explore the mechanism by which this alkaloid induces apoptosis in human ovarian cancer (CAOV-3) cells in vitro from 24 to 72 h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells. Cytotoxicity was assessed via the cell viability assay and changes in cell morphology were observed via the acridine orange/propidium iodide (AO/PI) assay. The involvement of apoptotic pathways was evaluated through caspase analysis and multiple cytotoxicity assays. Meanwhile, early and late apoptotic events via the Annexin V-FITC and DNA laddering assays, respectively. The mechanism of apoptosis was explored at the molecular level by evaluating the expression of specific genes and proteins. In addition, the proliferation of CAOV-3-cells treated with cleistopholine was analysed using the cell cycle arrest assay. The IC50 of cleistopholine (61.4 µM) was comparable with that of the positive control cisplatin (62.8 µM) at 24 h of treatment. Apoptos is was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24 h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48 h, indicating DNA fragmentation in the final phase of apoptosis. The mitochondria participated in the process by stimulating the intrinsic pathway via caspase 9 with a reduction in mitochondrial membrane potential (MMP) and an increase in cytochrome c release. Cell death was further validated through the mRNA and protein overexpression of Bax, caspase 3 and caspase 9 in the treated cells compared with the untreated cells. In contrast, Bcl-2, Hsp70 and survivin decreased in expression upon cleistopholine treatment. Cell cycle was arrested at the G0/G1 phase and cell population percentage significantly increased to 43.5%, 45.4% and 54.3% in time-dependent manner in the cleistopholine-treated CAOV-3 cells compared with the untreated cells at 24, 48 and 72 h respectively. The current study indicated that cleistopholine can be a potential candidate as a new drug to treat ovarian cancer disease. Copyright © 2016 Elsevier GmbH. All rights reserved.

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus).

PubMed

Parthipan, Sivashanmugam; Selvaraju, Sellappan; Somashekar, Lakshminarayana; Kolte, Atul P; Arangasamy, Arunachalam; Ravindra, Janivara Parameswaraiah

2015-08-01

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at -80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy+TRIzol and PureLink+TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800-900 ng/30-40 × 10(6)) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50-2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa. Copyright © 2015 Elsevier Inc. All rights reserved.

Mechanisms of Cochlear Stimulation Through the Round Window

NASA Astrophysics Data System (ADS)

Lukashkin, Andrei N.; Weddell, Thomas; Russell, Ian J.

2011-11-01

The round window membrane (RW) functions as a pressure relief valve in conventional hearing allowing structures of the middle ear to move. Investigations in recent years have shown that middle ear implants can be used to stimulate the cochlea via the RW. Isolated clinical uses of this technique have been applied but more thorough theoretical and empirical studies are required. Using guinea pigs as test subjects we have investigated physiological effects of RW stimulation using a simulation of active middle ear prosthesis, a cylindrical neodymium iron boron disk magnet placed upon the RW which can be stimulated by an electromagnetic coil positioned in close proximity to the magnet.

Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

PubMed Central

Elustondo, Pia A.; Angelova, Plamena R.; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y.; Pavlov, Evgeny V.

2013-01-01

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

PubMed

Elustondo, Pia A; Angelova, Plamena R; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y; Pavlov, Evgeny V

2013-01-01

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.

[L-glutamate-activated conductivity in the membrane of isolated pyramidal neurons of the rat hippocampus].

PubMed

Kiksin, N I; Tsyndrenko, A Ia

1985-01-01

Isolated pyramidal neurons from rat hippocampus were investigated under conditions of intracellular perfusion and voltage clamp using the method of rapid application of extracellular solution. It was found that the L-glutamate-activated current was carried by sodium and potassium ions with PK+/PNa+ ratio about 0.6. The dose-response relationship demonstrated one to one interaction between L-glutamate and membrane receptor with Kd value about 1.1 mmol/l.

VLDL metabolism in rats is affected by the concentration and source of dietary protein.

PubMed

Madani, Sihem; Prost, Josiane; Narce, Michel; Belleville, Jacques

2003-12-01

The present study was designed to determine if changes in dietary protein level and source are related to changes in VLDL lipid concentrations and VLDL binding by hepatic membranes and isolated hepatocytes. Male Wistar rats were fed cholesterol-free diets containing 10, 20 or 30 g/100 g casein or highly purified soybean protein for 4 wk. Hepatic, plasma and VLDL lipids, VLDL apo B-100 and VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane were determined. Increasing casein or soybean protein level (from 10 to 30 g/100 g) in the diet increased VLDL apo B-100, indicating an increase in the number of VLDL particles. VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane increased when the protein level increased from 10 to 20 g/100 g in the diet and decreased with 30 g/100 g protein, regardless of protein type. The dietary protein source did not affect plasma total cholesterol concentrations at any protein level. Feeding 20 g/100 g soybean protein compared with casein lowered plasma triglyceride concentrations and VLDL number as measured by decreased VLDL-protein, -phospholipid, -triglyceride, -cholesterol and -apo B-100. VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane were higher in rats fed soybean protein than those fed casein. The higher VLDL uptake could be responsible for the hypotriglyceridemia in rats fed soybean protein.

Isolating specific cell and tissue compartments from 3D images for quantitative regional distribution analysis using novel computer algorithms.

PubMed

Fenrich, Keith K; Zhao, Ethan Y; Wei, Yuan; Garg, Anirudh; Rose, P Ken

2014-04-15

Isolating specific cellular and tissue compartments from 3D image stacks for quantitative distribution analysis is crucial for understanding cellular and tissue physiology under normal and pathological conditions. Current approaches are limited because they are designed to map the distributions of synapses onto the dendrites of stained neurons and/or require specific proprietary software packages for their implementation. To overcome these obstacles, we developed algorithms to Grow and Shrink Volumes of Interest (GSVI) to isolate specific cellular and tissue compartments from 3D image stacks for quantitative analysis and incorporated these algorithms into a user-friendly computer program that is open source and downloadable at no cost. The GSVI algorithm was used to isolate perivascular regions in the cortex of live animals and cell membrane regions of stained spinal motoneurons in histological sections. We tracked the real-time, intravital biodistribution of injected fluorophores with sub-cellular resolution from the vascular lumen to the perivascular and parenchymal space following a vascular microlesion, and mapped the precise distributions of membrane-associated KCC2 and gephyrin immunolabeling in dendritic and somatic regions of spinal motoneurons. Compared to existing approaches, the GSVI approach is specifically designed for isolating perivascular regions and membrane-associated regions for quantitative analysis, is user-friendly, and free. The GSVI algorithm is useful to quantify regional differences of stained biomarkers (e.g., cell membrane-associated channels) in relation to cell functions, and the effects of therapeutic strategies on the redistributions of biomolecules, drugs, and cells in diseased or injured tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis.

PubMed Central

Chamberland, S; Bayer, A S; Schollaardt, T; Wong, S A; Bryan, L E

1989-01-01

Mechanisms of resistance to quinolones were characterized in Pseudomonas aeruginosa strains isolated after Tn5 insertional mutagenesis and in resistant strains that emerged during pefloxacin therapy of experimental aortic endocarditis. Quinolone resistance achieved in in vitro-selected mutants Qr-1 and Qr-2 was associated with cross-resistance to several groups of antimicrobial agents, including beta-lactams, tetracycline, and chloramphenicol. A significant reduction of norfloxacin uptake was also observed. After ether permeabilization of the cells, DNA synthesis of these two isolates was as susceptible to norfloxacin as DNA synthesis of the parent strain (PAO1). These results indicate that alteration of outer membrane permeability is the primary determinant of resistance in these isolates. This altered cell permeability was correlated with reduction of outer membrane protein G (25.5 kilodaltons) and loss of a 40-kilodalton outer membrane protein in strain Qr-1. Resistance to quinolones that emerged during experimental endocarditis therapy was associated with both modification of outer membrane permeability (decreased uptake of norfloxacin) and decreased susceptibility of DNA synthesis to norfloxacin. Resistance was limited to quinolones and chloramphenicol. For these strains, norfloxacin inhibitory doses (50%) for DNA synthesis were identical to the drug MICs, suggesting that despite the identification of a permeability change, perhaps due to changes of lipopolysaccharide, the alteration of the quinolone intracellular target(s) susceptibility constitutes the primary determinant of resistance. Also, two distinct levels of norfloxacin resistance of DNA synthesis were found in these isolates, indicating that at least two distinct alterations of the drug target(s) are possible in P. aeruginosa. Images PMID:2502066

Femtosecond laser-induced cell-cell surgical attachment.

PubMed

Katchinskiy, Nir; Godbout, Roseline; Goez, Helly R; Elezzabi, Abdulhakem Y

2014-04-01

Laser-induced cell-cell surgical attachment using femtosecond laser pulses is reported. We have demonstrated the ability to attach single cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength delivered from a Ti:Sapphire laser. To check that the cells did not go through a cell-fusion process, a fluorescent dye Calcein AM was used to verify that the fluorescent dye did not migrate from a dyed cell to a non-dyed cell. The mechanical integrity of the attached joint was assessed using an optical tweezer. Attachment of cells was performed without the induction of cell-cell fusion, with attachment efficiency of 95%, and while preserving the cells' viability. Cell-cell attachment was achieved by delivery of one to two trains of femtosecond laser pulses lasting 15 ms each. Laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane. The inner cell membrane remained intact during the attachment procedure, and isolation of the cells' cytoplasm from the surrounding medium was obtained. A strong physical attachment between the cells was obtained due to the bonding of the membranes' ionized phospholipid molecules and the formation of a joint cellular membrane at the connection point. The cellular attachment technique, femtosecond laser-induced cell-cell surgical attachment, can potentially provide a platform for the creation of engineered tissue and cell cultures. © 2014 Wiley Periodicals, Inc.

Properties of Fiber Cell Plasma Membranes Isolated from the Cortex and Nucleus of the Porcine Eye Lens

PubMed Central

Mainali, Laxman; Raguz, Marija; O’Brien, William J.; Subczynski, Witold K.

2012-01-01

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region. PMID:22326289

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine.

PubMed

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W; Cai, Jiye

2014-11-07

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

NASA Astrophysics Data System (ADS)

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

2014-10-01

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Sulzner, Michael; Egelseer, Eva; Norton, Cynthia F.; Hochstein, Lawrence I.

1993-01-01

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk-proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Comparison of Membrane ATPases from Extreme Halophiles Isolated from Ancient Salt Deposits

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Sulzner, Michael; Egelseer, Eva; Norton, Cynthia F.; Hochstein, Lawrence I.

1993-01-01

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to sub unit composition. enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

PubMed

McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

2016-08-06

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

Yeast Membrane Vesicles: Isolation and General Characteristics1

PubMed Central

Christensen, Michael S.; Cirillo, Vincent P.

1972-01-01

Yeast membrane vesicles are formed when packed yeast are ground manually in a porcelain mortar and pestle with glass beads (0.2 mm diameter). These vesicles can be separated from the other components of the grinding mixture by a combination of centrifugation steps and elution from a column of the same glass beads (0.2 mm diameter). Isolated vesicles are osmotically sensitive, contain cytoplasmic components, and have energy-independent transport function. They are unable to metabolize glucose, but have respiratory function which is thought to be associated with intravesicular mitochondria. Invertase and oligomycin-insensitive adenosine triphosphatase are present in lysed vesicle preparations, and the appropriateness of these enzyme activities as membrane markers is discussed. Images PMID:4337848

Arrays of membrane isolated yttrium-barium-copper-oxide kinetic inductance bolometers

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

Lindeman, M. A., E-mail: mark.a.lindeman@jpl.nasa.gov; Bonetti, J. A.; Bumble, B.

We are developing of arrays of membrane isolated resonator-bolometers, each with a kinetic inductance device (KID) to measure the temperature of the membrane. The KIDs are fabricated out of the high temperature superconductor YBCO to allow operation at relatively high temperatures. The bolometers are designed to offer higher sensitivity than sensors operating at 300 K, but they require less expensive and lighter weight cooling than even more sensitive conventional superconducting detectors operating at lower temperatures. The bolometer arrays are applicable as focal planes in infrared imaging spectrometers, such as for planetary science missions or earth observing satellites. We describe the devicesmore » and present measurements of their sensitivity.« less

Process for manufacture of semipermeable silicon nitride membranes

DOEpatents

Galambos, Paul Charles; Shul, Randy J.; Willison, Christi Gober

2003-12-09

A new class of semipermeable membranes, and techniques for their fabrication, have been developed. These membranes, formed by appropriate etching of a deposited silicon nitride layer, are robust, easily manufacturable, and compatible with a wide range of silicon micromachining techniques.

Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer☆

PubMed Central

Da Costa, Lydie; Suner, Ludovic; Galimand, Julie; Bonnel, Amandine; Pascreau, Tiffany; Couque, Nathalie; Fenneteau, Odile; Mohandas, Narla

2016-01-01

Inherited red blood cell (RBC) membrane disorders, such as hereditary spherocytosis, elliptocytosis and hereditary ovalocytosis, result from mutations in genes encoding various RBC membrane and skeletal proteins. The RBC membrane, a composite structure composed of a lipid bilayer linked to a spectrin/actin-based membrane skeleton, confers upon the RBC unique features of deformability and mechanical stability. The disease severity is primarily dependent on the extent of membrane surface area loss. RBC membrane disorders can be readily diagnosed by various laboratory approaches that include RBC cytology, flow cytometry, ektacytometry, electrophoresis of RBC membrane proteins and genetics. The reference technique for diagnosis of RBC membrane disorders is the osmotic gradient ektacytometry. However, in spite of its recognition as the reference technique, this technique is rarely used as a routine diagnosis tool for RBC membrane disorders due to its limited availability. This may soon change as a new generation of ektacytometer has been recently engineered. In this review, we describe the workflow of the samples shipped to our Hematology laboratory for RBC membrane disorder analysis and the data obtained for a large cohort of French patients presenting with RBC membrane disorders using a newly available version of the ektacytomer. PMID:26603718

Separation of milk fat globules via microfiltration: Effect of diafiltration media and opportunities for stream valorization.

PubMed

Jukkola, A; Partanen, R; Rojas, O J; Heino, A

2016-11-01

Milk fat globule membranes (MFGM) sourced in buttermilk have gained recent interest given their nutritional value and functional properties. However, production of isolated MFGM has been challenging given their size similarity with casein micelles, which limits attempts toward fractionation by size exclusion techniques. Therefore, the hypothesis underpinning this study is that the removal of proteins from cream before butter-making facilitates MFGM isolation. As such, milk fat globules were separated from raw whole milk via microfiltration (1.4-µm pore diameter and 0.005-m 2 filtration surface area) by using 3 diafiltration media; namely, skim milk ultrafiltration permeate, saline, and water. Their effects on the stability of the milk fat globules and protein permeation was elucidated. Whereas a substantial reduction in protein concentration was achieved with all diafiltration media (~90% reduction), water and saline produced negligible membrane fouling with better filtration performance. Moreover, diafiltration with skim milk ultrafiltration permeate exhibited reduced permeate flux. Colloidal stability of the resultant milk decreased with all diafiltration solutions due to changing composition and reduced apparent viscosity. Overall, microfiltration was found to be an efficient method for separation of milk fat globules from whole milk, leading to increased MFGM fragment concentration in buttermilk dry matter, thus making it more suitable for industrial utilization. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A novel fenestration technique for abdominal aortic dissection membranes using a combination of a needle re-entry catheter and the "cheese-wire" technique.

PubMed

Kos, Sebastian; Gürke, Lorenz; Jacob, Augustinus L

2011-12-01

This study was designed to demonstrate the applicability of a combined needle-based re-entry catheter and "cheese-wire" technique for fenestration of abdominal aortic dissection membranes. Four male patients (mean age: 65 years) with acute complicated aortic type B dissections were treated at our institution by fenestrating the abdominal aortic dissection membrane using a hybrid technique. This technique combined an initial membrane puncture with a needle-based re-entry catheter using a transfemoral approach. A guidewire was passed through the re-entry catheter and across the membrane. Using a contralateral transfemoral access, this guidewire was then snared, creating a through-and-through wire access. The membrane was then fenestrated using the cheese-wire maneuver. We successfully performed: (a) membrane puncture; (b) guidewire passage; (c) guidewire snaring; and (d) cheese-wire maneuver in all four cases. After this maneuver, decompression of the false lumen and acceptable arterial inflow into the true lumen was observed in all cases. The dependent visceral arteries were reperfused. In one case, portions of the fenestrated membrane occluded the common iliac artery, which was immediately and successfully stented. In another case, long-standing intestinal hypoperfusion before the fenestration resulted in reperfusion-related shock and intraoperative death of the patient. The described hybrid approach for fenestration of dissection membranes is technically feasible and may be established as a therapeutic method in cases with a complicated type B dissection.

Lytic agents, cell permeability, and monolayer penetrability.

PubMed

Salton, M R

1968-07-01

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20-30 % lipid and 50-75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2-3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

Electron microscopy of the nuclear membrane of Amoeba proteus.

PubMed

FRAJOLA, W J; GREIDER, M H; KOSTIR, W J

1956-07-25

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.

Water Reclamation Using a Ceramic Nanofiltration Membrane and Surface Flushing with Ozonated Water

PubMed Central

Hoang, Anh T.; Okuda, Tetsuji; Takeuchi, Haruka; Tanaka, Hiroaki; Nghiem, Long D.

2018-01-01

A new membrane fouling control technique using ozonated water flushing was evaluated for direct nanofiltration (NF) of secondary wastewater effluent using a ceramic NF membrane. Experiments were conducted at a permeate flux of 44 L/m2h to evaluate the ozonated water flushing technique for fouling mitigation. Surface flushing with clean water did not effectively remove foulants from the NF membrane. In contrast, surface flushing with ozonated water (4 mg/L dissolved ozone) could effectively remove most foulants to restore the membrane permeability. This surface flushing technique using ozonated water was able to limit the progression of fouling to 35% in transmembrane pressure increase over five filtration cycles. Results from this study also heighten the need for further development of ceramic NF membrane to ensure adequate removal of pharmaceuticals and personal care products (PPCPs) for water recycling applications. The ceramic NF membrane used in this study showed approximately 40% TOC rejection, and the rejection of PPCPs was generally low and highly variable. It is expected that the fouling mitigation technique developed here is even more important for ceramic NF membranes with smaller pore size and thus better PPCP rejection. PMID:29671797

Water Reclamation Using a Ceramic Nanofiltration Membrane and Surface Flushing with Ozonated Water.

PubMed

Fujioka, Takahiro; Hoang, Anh T; Okuda, Tetsuji; Takeuchi, Haruka; Tanaka, Hiroaki; Nghiem, Long D

2018-04-19

A new membrane fouling control technique using ozonated water flushing was evaluated for direct nanofiltration (NF) of secondary wastewater effluent using a ceramic NF membrane. Experiments were conducted at a permeate flux of 44 L/m²h to evaluate the ozonated water flushing technique for fouling mitigation. Surface flushing with clean water did not effectively remove foulants from the NF membrane. In contrast, surface flushing with ozonated water (4 mg/L dissolved ozone) could effectively remove most foulants to restore the membrane permeability. This surface flushing technique using ozonated water was able to limit the progression of fouling to 35% in transmembrane pressure increase over five filtration cycles. Results from this study also heighten the need for further development of ceramic NF membrane to ensure adequate removal of pharmaceuticals and personal care products (PPCPs) for water recycling applications. The ceramic NF membrane used in this study showed approximately 40% TOC rejection, and the rejection of PPCPs was generally low and highly variable. It is expected that the fouling mitigation technique developed here is even more important for ceramic NF membranes with smaller pore size and thus better PPCP rejection.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

PubMed Central

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

Mitochondrial Ceramide-Rich Macrodomains Functionalize Bax upon Irradiation

PubMed Central

Lee, Hyunmi; Rotolo, Jimmy A.; Mesicek, Judith; Penate-Medina, Tuula; Rimner, Andreas; Liao, Wen-Chieh; Yin, Xianglei; Ragupathi, Govind; Ehleiter, Desiree; Gulbins, Erich; Zhai, Dayong; Reed, John C.; Haimovitz-Friedman, Adriana; Fuks, Zvi; Kolesnick, Richard

2011-01-01

Background Evidence indicates that Bax functions as a “lipidic” pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates. Methodology/Principal Findings MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1–2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight “pore-forming” oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax. Conclusions/Significance Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo. PMID:21695182

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

PubMed

Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

2017-05-01

The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

Mycoplasma and associated bacteria isolated from ovine pink-eye.

PubMed

Langford, E V

1971-01-01

A mycoplasma was recovered from the untreated conjunctival membranes of nine sheep affected by Pink-eye. It was neither isolated from the conjunctiva of treated animals which were affected nor from the conjunctiva of normal animals either in contact or not in contact with affected animals. Bacteria found on normal conjunctival membranes were Neisseria ovis, Escherichia coli, Staphylococcus epidermididis, Streptococcus and Bacillus spp. Bacteria found in clinical cases of Pink-eye were N. ovis, E. coli, a Streptococcus and Pseudomonas spp.

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

PubMed

Thuleau, P; Ward, J M; Ranjeva, R; Schroeder, J I

1994-07-01

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Customizing model membranes and samples for NMR spectroscopic studies of complex membrane proteins.

PubMed

Sanders, C R; Oxenoid, K

2000-11-23

Both solution and solid state nuclear magnetic resonance (NMR) techniques for structural determination are advancing rapidly such that it is possible to contemplate bringing these techniques to bear upon integral membrane proteins having multiple transmembrane segments. This review outlines existing and emerging options for model membrane media for use in such studies and surveys the special considerations which must be taken into account when preparing larger membrane proteins for NMR spectroscopic studies.

Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.

PubMed

Nagaraj, V; Skillman, L; Li, D; Xie, Z; Ho, G

2017-07-01

Control of biofouling on seawater reverse osmosis (SWRO) membranes is a major challenge as treatments can be expensive, damage the membrane material and often biocides do not remove the polymers in which bacteria are embedded. Biological control has been largely ignored for biofouling control. The objective of this study was to demonstrate the effectiveness of xanthine oxidase enzyme against complex fouling communities and then identify naturally occurring bacterial strains that produce the free radical generating enzyme. Initially, 64 bacterial strains were isolated from different locations of the Perth Seawater Desalination Plant. In our preceding study, 25/64 isolates were selected from the culture collection as models for biofouling studies, based on their prevalence in comparison to the genomic bacterial community. In this study, screening of these model strains was performed using a nitroblue tetrazolium assay in the presence of hypoxanthine as substrate. Enzyme activity was measured by absorbance. Nine of 25 strains tested positive for xanthine oxidase production, of which Exiguobacterium from sand filters and Microbacterium from RO membranes exhibited significant levels of enzyme production. Other genera that produced xanthine oxidase were Marinomonas, Pseudomonas, Bacillus, Pseudoalteromonas and Staphylococcus. Strain variations were observed between members of the genera Microbacterium and Bacillus. Xanthine oxidase, an oxidoreductase enzyme that generates reactive oxygen species, is endogenously produced by many bacterial species. In this study, production of the enzyme by bacterial isolates from a full-scale desalination plant was investigated for potential use as biological control of membrane fouling in seawater desalination. We have previously demonstrated that free radicals generated by a commercially available xanthine oxidase in the presence of a hypoxanthine substrate, effectively dispersed biofilm polysaccharides on industrially fouled membranes. Bacterial xanthine oxidase production in the presence of hypoxanthine may prove to be a cost effective, in situ method for alleviation of fouling. © 2017 The Society for Applied Microbiology.

Free Flow Zonal Electrophoresis for Fractionation of Plant Membrane Compartments Prior to Proteomic Analysis.

PubMed

Barkla, Bronwyn J

2018-01-01

Free flow zonal electrophoresis (FFZE) is a versatile, reproducible, and potentially high-throughput technique for the separation of plant organelles and membranes by differences in membrane surface charge. It offers considerable benefits over traditional fractionation techniques, such as density gradient centrifugation and two-phase partitioning, as it is relatively fast, sample recovery is high, and the method provides unparalleled sample purity. It has been used to successfully purify chloroplasts and mitochondria from plants but also, to obtain highly pure fractions of plasma membrane, tonoplast, ER, Golgi, and thylakoid membranes. Application of the technique can significantly improve protein coverage in large-scale proteomics studies by decreasing sample complexity. Here, we describe the method for the fractionation of plant cellular membranes from leaves by FFZE.

Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

PubMed

Kiechle, F L; Sykes, E; Artiss, J D

1995-01-01

Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane

PubMed Central

Binte Atique, Fahmida; Ahmed, Kazi Tahsin; Asaduzzaman, S. M.; Hasan, Kazi Nadim

2013-01-01

Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D10) values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4 × 102 to 1.2 × 105 cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%). The D10 values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD) of 11.4 KGy for a bioburden level of 1000. To compare the differences, D10 values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes. PMID:24063009

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton.

PubMed

Basu, Avik; Harper, Sandra; Pesciotta, Esther N; Speicher, Kaye D; Chakrabarti, Abhijit; Speicher, David W

2015-10-14

Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria

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

Almukhtar, H.; Garle, M.J.; Smith, P.A.

2016-08-15

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques. Simvastatin, but not pravastatin, produced amore » slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10 μM) and the complex III inhibitor myxothiazol (10 μM), or a combination of the two. The complex III inhibitor antimycin A (10 μM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10 μM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone. In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel. - Highlights: • Simvastatin produces a relaxation of the porcine coronary artery. • This relaxation is inhibited by mitochondrial complex inhibitors. • Simvastatin alters mitochondrial membrane potential in intact blood vessels. • Simvastatin inhibits calcium influx in smooth muscle cells, prevented by a mitochondrial inhibitor. • Simvastatin-induced relaxations are dependent upon mitochondrial activity.« less

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

PubMed

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

2017-11-06

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

Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients.

PubMed

Gholipourmalekabadi, M; Bandehpour, M; Mozafari, M; Hashemi, A; Ghanbarian, H; Sameni, M; Salimi, M; Gholami, M; Samadikuchaksaraei, A

2015-11-01

Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

Gold/silver coated nanoporous ceramic membranes: a new substrate for SERS studies

NASA Astrophysics Data System (ADS)

Kassu, A.; Robinson, P.; Sharma, A.; Ruffin, P. B.; Brantley, C.; Edwards, E.

2010-08-01

Surface Enhanced Raman Scattering (SERS) is a recently discovered powerful technique which has demonstrated sensitivity and selectivity for detecting single molecules of certain chemical species. This is due to an enhancement of Raman scattered light by factors as large as 1015. Gold and Silver-coated substrates fabricated by electron-beam lithography on Silicon are widely used in SERS technique. In this paper, we report the use of nanoporous ceramic membranes for SERS studies. Nanoporous membranes are widely used as a separation membrane in medical devices, fuel cells and other studies. Three different pore diameter sizes of commercially available nanoporous ceramic membranes: 35 nm, 55nm and 80nm are used in the study. To make the membranes SERS active, they are coated with gold/silver using sputtering techniques. We have seen that the membranes coated with gold layer remain unaffected even when immersed in water for several days. The results show that gold coated nanoporous membranes have sensitivity comparable to substrates fabricated by electron-beam lithography on Silicon substrates.

Cell-cell interactions of isolated and cultured oligodendrocytes: formation of linear occluding junctions and expression of peculiar intramembrane particles.

PubMed

Massa, P T; Szuchet, S; Mugnaini, E

1984-12-01

Oligodendrocytes were isolated from lamb brain. Freshly isolated cells and cultured cells, either 1- to 4-day-old unattached or 1- to 5-week-old attached, were examined by thin section and freeze-fracture electron microscopy. Freeze-fracture of freshly isolated oligodendrocytes showed globular and elongated intramembrane particles similar to those previously described in oligodendrocytes in situ. Enrichment of these particles was seen at sites of inter-oligodendrocyte contact. Numerous gap junctions and scattered linear tight junctional arrays were apparent. Gap junctions were connected to blebs of astrocytic plasma membrane sheared off during isolation, whereas tight junctions were facing extracellular space or blebs of oligodendrocytic plasma membrane. Thin sections of cultured, unattached oligodendrocytes showed rounded cell bodies touching one another at points without forming specialized cell junctions. Cells plated on polylysine-coated aclar dishes attached, emanated numerous, pleomorphic processes, and expressed galactocerebroside and myelin basic protein, characteristic markers for oligodendrocytes. Thin sections showed typical oligodendrocyte ultrastructure but also intermediate filaments not present in unattached cultures. Freeze-fracture showed intramembrane particles similar to but more numerous, and with a different fracture face repartition, than those seen in oligodendrocytes, freshly isolated or in situ. Gap junctions were small and rare. Apposed oligodendrocyte plasma membrane formed linear tight junctions which became more numerous with time in culture. Thus, cultured oligodendrocytes isolated from ovine brains develop and maintain features characteristic of mature oligodendrocytes in situ and can be used to explore formation and maintenance of tight junctions and possibly other classes of cell-cell interactions important in the process of myelination.

Application of Fourier transform infrared spectroscopy and chemometrics for differentiation of Salmonella enterica serovar Enteritidis phage types.

PubMed

Preisner, Ornella; Guiomar, Raquel; Machado, Jorge; Menezes, José Cardoso; Lopes, João Almeida

2010-06-01

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm(-1). Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.

Thymocyte plasma membrane of the rainbow trout, Salmo gairdneri: Associated immunoglobulin and heteroantigens

USGS Publications Warehouse

Warr, G.W.; DeLuca, D.; Anderson, D.P.

1983-01-01

1. Thymic lymphocytes of the rainbow trout, S. gairdneri were disrupted and a plasma membrane containing fraction isolated by differential and buoyant density centrifugation.2. Radioiodine introduced into the membrane by the lactoperoxidase catalyzed reaction and immunoglobulin (identified by radioimmunoassay with monoclonal antibody) both copurified in the plasma membrane fraction.3. Rabbit antibody raised to the plasma membrane fraction showed a strong reaction with trout lymphocytes in immunofluorescence, was mitogenic for trout lymphocytes, and recognized lymphocyte membrane heteroantigens of molecular weight > 70,000 in the thymus and 45,000–95,000 in the head kidney.

SNARE-mediated membrane fusion in autophagy

PubMed Central

Wang, Yongyao; Li, Linsen; Hou, Chen; Lai, Ying; Long, Jiangang; Liu, Jiankang; Zhong, Qing; Diao, Jiajie

2016-01-01

Autophagy, a conserved self-eating process for the bulk degradation of cytoplasmic materials, involves double-membrane autophagosomes formed when an isolation membrane emerges and their direct fusion with lysosomes for degradation. For the early biogenesis of autophagosomes and their later degradation in lysosomes, membrane fusion is necessary, although different sets of genes and autophagy-related proteins involved in distinct fusion steps have been reported. To clarify the molecular mechanism of membrane fusion in autophagy, to not only expand current knowledge of autophagy, but also benefit human health, this review discusses key findings that elucidate the unique membrane dynamics of autophagy. PMID:27422330

Antimicrobial sensitivity pattern of bacterial pathogens in urinary tract infections in South Delhi, India.

PubMed

Akhtar, Mohammad Shabib; Mohsin, Nehal; Zahak, Ahmad; Ain, Mohammad Ruhal; Pillai, P K; Kapur, Prem; Ahmad, Mohammad Zaki

2014-01-01

Seventy-four bacterial proven cases of urinary tract infections were studied, and identified by Mac Conkey agar and blood agar medium separately; all the isolates were subjected to antimicrobial sensitivity testing by Stokes technique. Ninty-six percent of total isolated organisms were found to be gram negative while remaining 4% were gram positive. Among gram negatives, E. coli and gram positive S. aureus were the most prevalent organisms. The percentage of gram negative isolates were as follows, E. coli (79.7%) followed by Klebsiella (9.5%), Pseudomonas, Acinetobacter were (2.7% each), Proteus constituted (1.4%). and among gram positive S. aureus (4%). The antibiotic resistance of identified organisms was carried out by disc-diffusion method with commercially available disc of thirteen antibiotics having different mode of actions such as inhibition of cell wall synthesis, membrane permeability alternatives, inhibition of protein synthesis and DNA synthesis inhibitors. Gram negatives showed more resistance to these antibiotics as compared to gram positive organisms. The most effective antibiotic for gram negative UTI isolates is amikacin showing 63% efficacy followed by Cefotaxime 55% efficacy, Amoxicillin and Ciprofloxacin with (49% each) efficacy. Among gram positives, Chloramphenicol, Co-trimoxazole, Gentamicin, Amikacin, Ciprofloxacin and Cefotaxime are most effective with (66.6% each) efficacy, then Ampicillin, Amoxicillin, Tetracycline and norfloxacin with (33.3% each) efficacy.

Interaction of actin filaments with the plasma membrane in Amoeba proteus: studies using a cell model and isolated plasma membrane.

PubMed

Kawakatsu, T; Kikuchi, A; Shimmen, T; Sonobe, S

2000-08-01

We prepared a cell model of Amoeba proteus by mechanical bursting to study the interaction between actin filaments (AFs) and plasma membrane (PM). The cell model prepared in the absence of Ca2+ showed remarkable contraction upon addition of ATP. When the model was prepared in the presence of Ca2+, the cytoplasmic granules formed an aggregate in the central region, having moved away from PM. Although this model showed contraction upon addition of ATP in the presence of Ca2+, less contraction was noted. Staining with rhodamine-phalloidin revealed association of AFs with PM in the former model, and a lesser amount of association in the latter model. The interaction between AFs and PM was also studied using the isolated PM. AFs were associated with PM isolated in the absence of Ca2+, but were not when Ca2+ was present. These results suggest that the interaction between AFs and PM is regulated by Ca2+.

[Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

PubMed

Venediktova, N I; Kuznetsov, K V; Gritsenko, E N; Gulidova, G P; Mironova, G D

2012-01-01

Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

PubMed Central

Owen, Peter; Salton, Milton R. J.

1977-01-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions. Images PMID:144722

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

PubMed

Owen, P; Salton, M R

1977-12-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Isolation of integrin-based adhesion complexes.

PubMed

Jones, Matthew C; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Robertson, Joseph; Paul, Nikki R; Ng, Daniel H J; Askari, Janet A; Humphries, Martin J

2015-03-02

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

Determination of collagen fibril size via absolute measurements of second-harmonic generation signals

NASA Astrophysics Data System (ADS)

Bancelin, Stéphane; Aimé, Carole; Gusachenko, Ivan; Kowalczuk, Laura; Latour, Gaël; Coradin, Thibaud; Schanne-Klein, Marie-Claire

2014-09-01

The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet’s membrane of a diabetic rat cornea.

A Protein Nanopore-Based Approach for Bacteria Sensing

NASA Astrophysics Data System (ADS)

Apetrei, Aurelia; Ciuca, Andrei; Lee, Jong-kook; Seo, Chang Ho; Park, Yoonkyung; Luchian, Tudor

2016-11-01

We present herein a first proof of concept demonstrating the potential of a protein nanopore-based technique for real-time detection of selected Gram-negative bacteria ( Pseudomonas aeruginosa or Escherichia coli) at a concentration of 1.2 × 108 cfu/mL. The anionic charge on the bacterial outer membrane promotes the electrophoretically driven migration of bacteria towards a single α-hemolysin nanopore isolated in a lipid bilayer, clamped at a negative electric potential, and followed by capture at the nanopore's mouth, which we found to be described according to the classical Kramers' theory. By using a specific antimicrobial peptide as a putative molecular biorecognition element for the bacteria used herein, we suggest that the detection system can combine the natural sensitivity of the nanopore-based sensing techniques with selective biological recognition, in aqueous samples, and highlight the feasibility of the nanopore-based platform to provide portable, sensitive analysis and monitoring of bacterial pathogens.

Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains of Porphyromonas gingivalis

PubMed Central

Sawada, Koichi; Kokeguchi, Susumu; Hongyo, Hiroshi; Sawada, Satoko; Miyamoto, Manabu; Maeda, Hiroshi; Nishimura, Fusanori; Takashiba, Shogo; Murayama, Yoji

1999-01-01

Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains. PMID:10531208

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

PubMed Central

MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

2008-01-01

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer.

PubMed

Da Costa, Lydie; Suner, Ludovic; Galimand, Julie; Bonnel, Amandine; Pascreau, Tiffany; Couque, Nathalie; Fenneteau, Odile; Mohandas, Narla

2016-01-01

Inherited red blood cell (RBC) membrane disorders, such as hereditary spherocytosis, elliptocytosis and hereditary ovalocytosis, result from mutations in genes encoding various RBC membrane and skeletal proteins. The RBC membrane, a composite structure composed of a lipid bilayer linked to a spectrin/actin-based membrane skeleton, confers upon the RBC unique features of deformability and mechanical stability. The disease severity is primarily dependent on the extent of membrane surface area loss. RBC membrane disorders can be readily diagnosed by various laboratory approaches that include RBC cytology, flow cytometry, ektacytometry, electrophoresis of RBC membrane proteins and genetics. The reference technique for diagnosis of RBC membrane disorders is the osmotic gradient ektacytometry. However, in spite of its recognition as the reference technique, this technique is rarely used as a routine diagnosis tool for RBC membrane disorders due to its limited availability. This may soon change as a new generation of ektacytometer has been recently engineered. In this review, we describe the workflow of the samples shipped to our Hematology laboratory for RBC membrane disorder analysis and the data obtained for a large cohort of French patients presenting with RBC membrane disorders using a newly available version of the ektacytomer. Copyright © 2015 Elsevier Inc. All rights reserved.

Recent progress in the applications of layer-by-layer assembly to the preparation of nanostructured ion-rejecting water purification membranes.

PubMed

Sanyal, Oishi; Lee, Ilsoon

2014-03-01

Reverse osmosis (RO) and nanofiltration (NF) are the two dominant membrane separation processes responsible for ion rejection. While RO is highly efficient in removal of ions it needs a high operating pressure and offers very low selectivity between ions. Nanofiltration on the other hand has a comparatively low operating pressure and most commercial membranes offer selectivity in terms of ion rejection. However in many nanofiltration operations rejection of monovalent ions is not appreciable. Therefore a high flux high rejection membrane is needed that can be applied to water purification systems. One such alternative is the usage of polyelectrolyte multilayer membranes that are prepared by the deposition of alternately charged polyelectrolytes via layer-by-layer (LbL) assembly method. LbL is one of the most common self-assembly techniques and finds application in various areas. It has a number of tunable parameters like deposition conditions, number of bilayers deposited etc. which can be manipulated as per the type of application. This technique can be applied to make a nanothin membrane skin which gives high rejection and at the same time allow a high water flux across it. Several research groups have applied this highly versatile technique to prepare membranes that can be employed for water purification. Some of these membranes have shown better performance than the commercial nanofiltration and reverse osmosis membranes. These membranes have the potential to be applied to various different aspects of water treatment like water softening, desalination and recovery of certain ions. Besides the conventional method of LbL technique other alternative methods have also been suggested that can make the technique fast, more efficient and thereby make it more commercially acceptable.

Vascularization and Cellular Isolation Potential of a Novel Electrospun Cell Delivery Vehicle

PubMed Central

Krishnan, Laxminarayanan; Touroo, Jeremy; Reed, Robert; Boland, Eugene; Hoying, James B.; Williams, Stuart K.

2014-01-01

A clinical need exists for a cell delivery device that supports long term cell viability, cell retention within the device and retrieval of delivered cells if necessary. Previously, cell isolation devices have been based on hollow fiber membranes, porous polymer scaffolds, alginate systems, or micro-machined membranes. We present the development and characterization of a novel dual porosity electrospun membrane based device, which supports cellular infiltration and vascularization of its outer porous layer and maintains cellular isolation within a lumen bounded by an inner low porosity layer. Electrospinning conditions were initially established to support electrospun fiber deposition onto nonconductive silicone surfaces. With these parameters established, devices for in vivo evaluations were produced using nylon as a nonconductive scaffold for deposition of dual porosity electrospun fibers. The outer porous layer supported the development of a penetrating microcirculation and the membrane supported the transfer of insulin from encapsulated sustained release pellets for four weeks. Viable cells implanted within the device could be identified after two weeks of implantation. Through the successful demonstration of survival and cellular isolation of human epithelial cells within the implanted devices and the ability to use the device to deliver insulin, we have established the utility of this device toward localized cell transplantation. The Cell Delivery Device establishes a platform to test the feasibility of approaches to cell dose control and cell localization at the site of implantation in the clinical use of modified autologous or allogeneic cells. PMID:23913805

Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

PubMed

Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

2014-01-01

In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin.

PubMed

Mata-Cabana, Alejandro; Florencio, Francisco J; Lindahl, Marika

2007-11-01

Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.

Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion.

PubMed

Brejchová, Jana; Sýkora, Jan; Dlouhá, Kateřina; Roubalová, Lenka; Ostašov, Pavel; Vošahlíková, Miroslava; Hof, Martin; Svoboda, Petr

2011-12-01

Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade. Copyright © 2011 Elsevier B.V. All rights reserved.

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

NASA Technical Reports Server (NTRS)

Hendrix, D. L.

1978-01-01

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

High affinity 3H-Phe uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBMVw)

USDA-ARS?s Scientific Manuscript database

Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to s...

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

NASA Astrophysics Data System (ADS)

Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

2013-08-01

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Characterizing nanoscale topography of the aortic heart valve basement membrane for tissue engineering heart valve scaffold design.

PubMed

Brody, Sarah; Anilkumar, Thapasimuthu; Liliensiek, Sara; Last, Julie A; Murphy, Christopher J; Pandit, Abhay

2006-02-01

A fully effective prosthetic heart valve has not yet been developed. A successful tissue-engineered valve prosthetic must contain a scaffold that fully supports valve endothelial cell function. Recently, topographic features of scaffolds have been shown to influence the behavior of a variety of cell types and should be considered in rational scaffold design and fabrication. The basement membrane of the aortic valve endothelium provides important parameters for tissue engineering scaffold design. This study presents a quantitative characterization of the topographic features of the native aortic valve endothelial basement membrane; topographical features were measured, and quantitative data were generated using scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and light microscopy. Optimal conditions for basement membrane isolation were established. Histological, immunohistochemical, and TEM analyses following decellularization confirmed basement membrane integrity. SEM and AFM photomicrographs of isolated basement membrane were captured and quantitatively analyzed. The basement membrane of the aortic valve has a rich, felt-like, 3-D nanoscale topography, consisting of pores, fibers, and elevations. All features measured were in the sub-100 nm range. No statistical difference was found between the fibrosal and ventricular surfaces of the cusp. These data provide a rational starting point for the design of extracellular scaffolds with nanoscale topographic features that mimic those found in the native aortic heart valve basement membrane.

Characterizing Nanoscale Topography of the Aortic Heart Valve Basement Membrane for Tissue Engineering Heart Valve Scaffold Design

PubMed Central

BRODY, SARAH; ANILKUMAR, THAPASIMUTHU; LILIENSIEK, SARA; LAST, JULIE A.; MURPHY, CHRISTOPHER J.; PANDIT, ABHAY

2016-01-01

A fully effective prosthetic heart valve has not yet been developed. A successful tissue-engineered valve prosthetic must contain a scaffold that fully supports valve endothelial cell function. Recently, topographic features of scaffolds have been shown to influence the behavior of a variety of cell types and should be considered in rational scaffold design and fabrication. The basement membrane of the aortic valve endothelium provides important parameters for tissue engineering scaffold design. This study presents a quantitative characterization of the topographic features of the native aortic valve endothelial basement membrane; topographical features were measured, and quantitative data were generated using scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and light microscopy. Optimal conditions for basement membrane isolation were established. Histological, immunohistochemical, and TEM analyses following decellularization confirmed basement membrane integrity. SEM and AFM photomicrographs of isolated basement membrane were captured and quantitatively analyzed. The basement membrane of the aortic valve has a rich, felt-like, 3-D nanoscale topography, consisting of pores, fibers, and elevations. All features measured were in the sub-100 nm range. No statistical difference was found between the fibrosal and ventricular surfaces of the cusp. These data provide a rational starting point for the design of extracellular scaffolds with nanoscale topographic features that mimic those found in the native aortic heart valve basement membrane. PMID:16548699

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

PubMed

Battle, Katrina N; Jackson, Joshua M; Witek, Małgorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A

2014-03-21

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

Label-free evanescent microscopy for membrane nano-tomography in living cells.

PubMed

Bon, Pierre; Barroca, Thomas; Lévèque-Fort, Sandrine; Fort, Emmanuel

2014-11-01

We show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells. Readily implementable on a standard inverted microscope, this technique enables full-field and real-time tracking of membrane processes without labeling and thus signal fading. In addition, we demonstrate that the membrane/interface distance can be retrieved with 10 nm precision using a multilayer Fresnel model. We apply this nano-axial tomography of living cell membranes to retrieve quantitative information on membrane invagination dynamics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Transport of Zn(OH)4(-2) ions across a polyolefin microporous membrane

NASA Astrophysics Data System (ADS)

Krejci, Ivan; Vanysek, Peter; Trojanek, Antonin

1993-04-01

Transport of ZN(OH)4(2-) ions through modified microporous polypropylene membranes (Celgard 3401, 350140) was studied using polarography and conductometry. Soluble Nafion as an ion exchange modifying agent was applied to the membrane by several techniques. The influence of Nafion and a surfactant on transport of zinc ions through the membrane was studied. A relationship between membrane impedance and the rate of Zn(OH)4(2-) transport was found. The found correlation between conductivity, ion permeability and Nafion coverage suggests a suitable technique of membrane preparation to obtain desired zinc ion barrier properties.

Shedding light on the puzzle of drug-membrane interactions: Experimental techniques and molecular dynamics simulations.

PubMed

Lopes, Daniela; Jakobtorweihen, Sven; Nunes, Cláudia; Sarmento, Bruno; Reis, Salette

2017-01-01

Lipid membranes work as barriers, which leads to inevitable drug-membrane interactions in vivo. These interactions affect the pharmacokinetic properties of drugs, such as their diffusion, transport, distribution, and accumulation inside the membrane. Furthermore, these interactions also affect their pharmacodynamic properties with respect to both therapeutic and toxic effects. Experimental membrane models have been used to perform in vitro assessment of the effects of drugs on the biophysical properties of membranes by employing different experimental techniques. In in silico studies, molecular dynamics simulations have been used to provide new insights at an atomistic level, which enables the study of properties that are difficult or even impossible to measure experimentally. Each model and technique has its advantages and disadvantages. Hence, combining different models and techniques is necessary for a more reliable study. In this review, the theoretical backgrounds of these (in vitro and in silico) approaches are presented, followed by a discussion of the pharmacokinetic and pharmacodynamic properties of drugs that are related to their interactions with membranes. All approaches are discussed in parallel to present for a better connection between experimental and simulation studies. Finally, an overview of the molecular dynamics simulation studies used for drug-membrane interactions is provided. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

PubMed

Szamel, M; Goppelt, M; Resch, K

1985-12-19

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Thermodynamics and Mechanics of Membrane Curvature Generation and Sensing by Proteins and Lipids

PubMed Central

Baumgart, Tobias; Capraro, Benjamin R.; Zhu, Chen; Das, Sovan L.

2014-01-01

Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature. We then survey several mechanisms that are assumed to underlie membrane curvature sensing and generation. Finally, we discuss relatively simple thermodynamic/mechanical models that allow quantitative interpretation of experimental observations. PMID:21219150

Mini-Scale Isolation and Preparation of Plasma Membrane Proteins from Potato Roots for LC/MS Analysis.

PubMed

Jozefowicz, Anna M; Matros, Andrea; Witzel, Katja; Mock, Hans-Peter

2018-01-01

Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30 g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5 g and allows studying the plasma membrane proteome by LC-MS.

ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

PubMed Central

Chauveau, J.; Moulé, Y.; Rouiller, C.; Schneebeli, J.

1962-01-01

Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry. PMID:13878497

Recycling of end-of-life reverse osmosis membranes by oxidative treatment: a technical evaluation.

PubMed

Coutinho de Paula, Eduardo; Gomes, Júlia Célia Lima; Amaral, Míriam Cristina Santos

2017-07-01

The adverse impacts caused by the disposal of thousands of tonnes per annum of reverse osmosis (RO) membranes modules have grown dramatically around the world. The objective of this study was to evaluate the technical feasibility of recycling by chemical oxidation of end-of-life RO membranes for applications in other separation processes with specifications less rigorous. The recycling technique consisted in to cause a membrane exposition with oxidant solutions in order to remove its aromatic polyamide layer and subsequent conversion to a porous membrane. The recycling technique was evaluated by water permeability and salt rejection tests before and after the oxidative treatments. Initially, membranes' chemical cleaning and pretreatment procedures were assessed. Among factors evaluated, the oxidizing agent, its concentration and pH, associated with the oxidative treatment time, showed important influence on the oxidation of the membranes. Results showed that sodium hypochlorite and potassium permanganate are efficient agents for the membrane recycling. The great increased permeability and decreased salt rejection indicated changes on membranes' selective properties. Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), and contact angle characterization techniques revealed marked changes on the main membranes' physical-chemical properties, such as morphology, roughness and hydrophobicity. Reuse of produced effluents and fouling tendency of recycled membranes were also evaluated.

Assessing heterogeneity of peroxisomes: isolation of two subpopulations from rat liver.

PubMed

Islinger, Markus; Abdolzade-Bavil, Afsaneh; Liebler, Sven; Weber, Gerhardt; Völkl, Alfred

2012-01-01

Peroxisomes exhibit a heterogeneous morphological appearance in rat liver tissue. In this respect, the isolation and subsequent biochemical characterization of peroxisome species from different subcellular prefractions should help to solve the question of whether peroxisomes indeed diverge into functionally specialized subgroups in one tissue. As a means to address this question, we provide a detailed separation protocol for the isolation of peroxisomes from both the light (LM-Po) and the heavy (HM-Po) mitochondrial prefraction for their subsequent comparative analysis. Both isolation strategies rely on centrifugation in individually adapted Optiprep gradients. In case of the heavy mitochondrial fraction, free flow electrophoresis is appended as an additional separation step to yield peroxisomes of sufficient purity. In view of their morphology, peroxisomes isolated from both fractions are surrounded by a continuous single membrane and contain a gray-opaque inner matrix. However, beyond this overall similar appearance, HM-Po exhibit a smaller average diameter, float at lower density, and show a more negative average membrane charge when compared to LM-Po.

Enhancement of algicidal properties of immobilized Bacillus methylotrophicus ZJU by coating with magnetic Fe₃O₄ nanoparticles and wheat bran.

PubMed

Sun, Pengfei; Hui, Cai; Wang, Sheng; Khan, Rashid Azim; Zhang, Qichun; Zhao, Yu-Hua

2016-01-15

Algicidal bacteria offer a promising option for killing cyanobacteria. In this study, a newly isolated strain of Bacillus methylotrophicus, ZJU, was used to control Microcystis aeruginosa. Analyses of relative reactive oxygen level, malondialdehyde content, superoxide dismutase activity, and fluorescence staining indicated that oxidative damage caused by the algicidal supernatant of strain ZJU mainly affected the cell membrane and consequently the membrane permeability and membrane potential of M. aeruginosa cells. Furthermore, an embedded immobilization technique was employed to improve the practical application of strain ZJU as an algicidal agent. On this basis, we proposed a novel concept of enhancing the algicidal properties of immobilized ZJU by adding Fe3O4 nanoparticles and wheat bran in the process of immobilization. Our studies showed that Fe3O4 nanoparticles conferred the immobilized bacteria with a magnetization of 30.87 emu/g, and this magnetization enabled efficient re-collection of the immobilized bacteria by magnetic means. Moreover, wheat bran endowed the immobilized bacteria with 10.34% higher algicidal activity than immobilized bacteria without wheat bran. The results indicate a novel concept of enhancing the algicidal property of bacteria against M. aeruginosa by adding Fe3O4 nanoparticles and wheat bran. Copyright © 2015 Elsevier B.V. All rights reserved.

Biological effects of Trichoderma harzianum peptaibols on mammalian cells.

PubMed

Peltola, Joanna; Ritieni, Alberto; Mikkola, Raimo; Grigoriev, Pavel A; Pócsfalvi, Gabriella; Andersson, Maria A; Salkinoja-Salonen, Mirja S

2004-08-01

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

PubMed Central

Peltola, Joanna; Ritieni, Alberto; Mikkola, Raimo; Grigoriev, Pavel A.; Pócsfalvi, Gabriella; Andersson, Maria A.; Salkinoja-Salonen, Mirja S.

2004-01-01

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen−1). The same exposure concentration depleted the boar sperm cells of NADH2. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C18 cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψm) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C18 cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS. PMID:15294840

Organelle-localized potassium transport systems in plants.

PubMed

Hamamoto, Shin; Uozumi, Nobuyuki

2014-05-15

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K(+) transport systems allowing K(+) to move across the membrane. K(+) transport systems in plant organelles act coordinately with the plasma membrane intrinsic K(+) transport systems to maintain cytosolic K(+) concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K(+) channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K(+) homeostasis of the cytoplasm. The initial electrophysiological measurements of K(+) transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K(+) transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K(+) transport system has been isolated from cyanobacteria, which may add to our understanding of K(+) flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K(+) transport proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

What's growing on General Practitioner's stethoscope?

PubMed

Carducci, A; Cargnelutti, M; Tassinari, F; Bizzarro, A; Cordio, G; Carletti, S; Maccarini, L; Pelissero, G

2016-01-01

Non-critical medical devices, as stethoscopes, have long been considered as vectors in microorganisms' transmission. Cleaning standards for non-critical medical equipment are often unclear. This study was designed to assess the attitude of General Practitioners (GPs) towards cleaning their stethoscope and the degree of microbiological contamination of doctor's instrument in outpatient setting. Observational, crossover study. A structured questionnaire was administered to GPs to test their knowledge about medical instrument's cleanliness recommendations and the surface of the diaphragm of their stethoscopes was analyzed for bacteriological isolates using mass spectrometry technique. Most of GPs declared they don't know cleaning recommendations for non-critical medical devices and a relevant bacterial growth was identified on the majority of the stethoscopes' membranes. Almost all microbiological isolates resulted typically found in cutaneous flora. We can't state that the GP's stethoscopes feature a risk of transmission for microbiological pathogens; anyway, because of the level of contamination we observed, cleaning recommendations to disinfect instruments on a regular basis should be better indicated.

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

PubMed Central

Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

2016-01-01

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

NASA Astrophysics Data System (ADS)

Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; Te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

2017-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

SNARE-mediated membrane fusion in autophagy.

PubMed

Wang, Yongyao; Li, Linsen; Hou, Chen; Lai, Ying; Long, Jiangang; Liu, Jiankang; Zhong, Qing; Diao, Jiajie

2016-12-01

Autophagy, a conserved self-eating process for the bulk degradation of cytoplasmic materials, involves double-membrane autophagosomes formed when an isolation membrane emerges and their direct fusion with lysosomes for degradation. For the early biogenesis of autophagosomes and their later degradation in lysosomes, membrane fusion is necessary, although different sets of genes and autophagy-related proteins involved in distinct fusion steps have been reported. To clarify the molecular mechanism of membrane fusion in autophagy, to not only expand current knowledge of autophagy, but also benefit human health, this review discusses key findings that elucidate the unique membrane dynamics of autophagy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-arthritic activity of 11-O-(4'-O-methyl galloyl)-bergenin and Crassula capitella extract in rats.

PubMed

El-Hawary, Seham S; Mohammed, Rabab; Abouzid, Sameh; Ali, Zeinab Y; Elwekeel, Ahlam

2016-06-01

Isolation and identification of phytochemicals of Crassula capitella (Thunberg), evaluation of the anti-arthritic potential of the extract and the major isolated compound; 11-O-(4'-O-methyl galloyl)-bergenin and underlying their mechanism on rat model of rheumatoid arthritis (RA). Different fractions were subjected to column chromatography giving fourteen compound identified by mass and NMR spectroscopic techniques. RA was induced by intraplantar injection of complete Freund's adjuvant into the right hind paw of rats. Influence of tested samples in comparable to methotrexate on paw oedema, body weight gain, serum diagnostic markers, cartilage and bone degeneration enzymes, pro-inflammatory mediators and oxidative stress biomarkers in arthritic rats. Fourteen phenolic compounds were isolated and identified for the first time from C. capitella. The major compound identified as 11-O-(4'-O-methyl galloyl)-bergenin. Treatment of arthritic rats with extract or 11-O-(4'-O-methyl galloyl)-bergenin with the tested doses can reduce the progression and severity of RA. Crassula capitella is a new natural and abundant source for 11-O-(4'-O-methyl galloyl)-bergenin for resolving chronic inflammatory diseases as RA through antioxidant, anti-inflammatory and membrane stabilizing mechanism. © 2016 Royal Pharmaceutical Society.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

PubMed Central

Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

2013-01-01

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm. PMID:23947891

Association between water and carbon dioxide transport in leaf plasma membranes: assessing the role of aquaporins.

PubMed

Zhao, Manchun; Tan, Hwei-Ting; Scharwies, Johannes; Levin, Kara; Evans, John R; Tyerman, Stephen D

2017-06-01

The role of some aquaporins as CO 2 permeable channels has been controversial. Low CO 2 permeability of plant membranes has been criticized because of unstirred layers and other limitations. Here we measured both water and CO 2 permeability (P os , P CO2 ) using stopped flow on plasma membrane vesicles (pmv) isolated from Pisum sativum (pea) and Arabidopsis thaliana leaves. We excluded the chemical limitation of carbonic anhydrase (CA) in the vesicle acidification technique for P CO2 using different temperatures and CA concentrations. Unstirred layers were excluded based on small vesicle size and the positive correlation between vesicle diameter and P CO2 . We observed high aquaporin activity (P os 0.06 to 0.22 cm s -1 ) for pea pmv based on all the criteria for their function using inhibitors and temperature dependence. Inhibitors of P os did not alter P CO2 . P CO2 ranged from 0.001 to 0.012 cm s -1 (mean 0.0079 + 0.0007 cm s -1 ) with activation energy of 30.2 kJ mol -1 . Intrinsic variation between pmv batches from normally grown or stressed plants revealed a weak (R 2  = 0.27) positive linear correlation between P os and P CO2 . Despite the low P CO2 , aquaporins may facilitate CO 2 transport across plasma membranes, but probably via a different pathway than for water. © 2016 John Wiley & Sons Ltd.

Laser light propagation in adipose tissue and laser effects on adipose cell membranes

NASA Astrophysics Data System (ADS)

Solarte, Efraín; Rebolledo, Aldo; Gutierrez, Oscar; Criollo, William; Neira, Rodrigo; Arroyave, José; Ramírez, Hugo

2006-01-01

Recently Neira et al. have presented a new liposuction technique that demonstrated the movement of fat from inside to outside of the cell, using a low-level laser device during a liposuction procedure with Ultrawet solution. The clinical observations, allowed this new surgical development, started a set of physical, histological and pharmacological studies aimed to determine the mechanisms involved in the observed fat mobilization concomitant to external laser application in liposuction procedures. Scanning and Transmission Electron Microscopy, studies show that the cellular arrangement of normal adipose tissue changes when laser light from a diode laser: 10 mW, 635 nm is applied. Laser exposures longer than 6 minutes cause the total destruction of the adipocyte panicles. Detailed observation of the adipose cells show that by short irradiation times (less than four minutes) the cell membrane exhibits dark zones, that collapse by longer laser exposures. Optical measurements show that effective penetration length depends on the laser intensity. Moreover, the light scattering is enhanced by diffraction and subsequent interference effects, and the tumescent solution produces a clearing of the tissue optical medium. Finally, isolate adipose cell observation show that fat release from adipocytes is a concomitant effect between the tumescent solution (adrenaline) and laser light, revealing a synergism which conduces to the aperture, and maybe the disruption, of the cell membrane. All these studies were consistent with a laser induced cellular process, which causes fat release from inside the adipocytes into the intercellular space, besides a strong modification of the cellular membranes.

High resolution labeling of cholinergic nerve terminals using a specific fully active biotinylated botulinum neurotoxin type A.

PubMed

Arribas, M; Blasi, J; Egea, G; Fariñas, I; Solsona, C; Marsal, J

1993-12-15

We report here on the synthesis and characterization of a fully active biotinylated derivative of the botulinum neurotoxin type A. Different ratios of biotin: botulinum toxin were tested to optimize derivatizing conditions and a ratio of 35:1 was selected for further experiments. The average number of biotin groups per toxin molecule was estimated to be 7.8, occurring at both heavy and light chains, and almost all externally located and easily accessible to recognition by streptavidin. The modified toxin retained its toxicity and its ability to interact with biological membranes. Apart from its suitability for detection in Western blots and in microtiter well plates, biotinylated botulinum toxin proved to be adequate for morphological labeling studies at both light and electron microscopy. Peroxidase histochemistry in cryostat sections of intoxicated rat hemidiaphragm muscles showed a distinct labeling of end-plates. Electron microscopy studies were performed on the electric organ of Torpedo marmorata using colloidal gold-conjugated streptavidin for detection. After intoxication of electric organ fragments with the modified toxin, gold labels were found associated with the presynaptic plasma membrane of nerve terminals and with the membrane of synaptic vesicles. Moreover, the distribution of biotinylated botulinum toxin binding sites over the membrane of synaptosomes isolated from the electric organ of Torpedo and their relationship with intramembrane particles were analyzed using the replica-staining label-fracture technique. It was found that the toxin is never associated with intramembrane particles.

Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

PubMed

Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

2016-01-01

Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

Identification and analysis of circulating exosomal microRNA in human body fluids.

PubMed

Lässer, Cecilia

2013-01-01

Exosomes are 40-100 nm sized vesicles released from cells when multivesicular bodies fuse with the plasma membrane. These vesicles take part in cell-to-cell communication by binding and signalling through membrane receptors on cells or by transferring proteins, RNA, and lipids into the cells. Exosomal RNA in body fluids, such as plasma and urine, has been associated with malignancies, making the exosomal RNA a potential biomarker for early detection of these diseases. This has increased the interest in the field of extracellular RNA and in particular, the interest in exosomal RNA.In this chapter, a well-established exosome isolation method is described, as well as how to characterize the isolated vesicles by electron microscopy. Furthermore, two types of RNA isolation methods are described with a focus on isolating RNA from body fluids, which can be more viscous than cell culture media.

The evolution of energy-transducing systems. Studies with an extremely halophilic archaebacterium

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga

1991-01-01

The halobacterial ATPase was labeled with C-14-dicyclohexylcarbodiimide and subunit 2 of the enzyme was prepared by electroelution. Subunit 2 was cleaved by several chemical and enzymatic procedures for further preparation of peptides. Immunoreactions (Western blotting) of halobacterial membranes were performed with an antiserum against subunit A of the vacuolar ATPase from Neurospora crassa. A 85 K band (subunit 1) from the membranes of H saccharovorum and from two halobacterial isolates, which were isolated from Permian salt sediments, reacted strongly with the antiserum. The ATPase from the latter isolates resembled the ATPase from H saccharovorum, but had a higher content of acidic amino acids. If it can be verified that the age of the bacterial isolates is in the same range as when deposition of salt occurred, an extremely interesting system for the study of evolutionary questions would be available, since the salt-embedded bacteria presumably did not undergo mutational and selectional events.

Color-Coded Organelles.

ERIC Educational Resources Information Center

McLaughlin, Esther; And Others

1994-01-01

Describes how red beets can be used to demonstrate a variety of membrane phenomena. Some of the activities include observation of vacuoles; vacuoles in intact cells; isolation of vacuoles in physiological studies; demonstration of membrane integrity; and demonstration of ion diffusion and active transport with purified vacuoles. (ZWH)

Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

PubMed

Pottosin, Igor; Velarde-Buendía, Ana María; Bose, Jayakumar; Fuglsang, Anja T; Shabala, Sergey

2014-06-01

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Chloride transport in functionally active phagosomes isolated from Human neutrophils

PubMed Central

Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

2012-01-01

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes. PMID:23089227

Chloride transport in functionally active phagosomes isolated from Human neutrophils.

PubMed

Aiken, Martha L; Painter, Richard G; Zhou, Yun; Wang, Guoshun

2012-12-15

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes. Copyright © 2012 Elsevier Inc. All rights reserved.

Long-lasting, distinct changes in central opioid receptor and urinary bladder functions in models of schizophrenia in rats.

PubMed

Kekesi, Orsolya; Tuboly, Gabor; Szucs, Maria; Birkas, Erika; Morvay, Zita; Benedek, Gyorgy; Horvath, Gyongyi

2011-07-01

Ketamine treatments and social isolation of rats reflect certain features of schizophrenia, among them altered pain sensitivity. To study the underlying mechanisms of these phenomena, rats were either housed individually or grouped for 33 days after weaning, and treated with either ketamine or saline for 14 days. After one month re-socialization, the urinary bladder capacity by ultrasound examination in the anesthetized animals, and changes of μ-opioid receptors by saturation binding experiments using a specific μ-opioid agonist [(3)H]DAMGO were determined. G-protein signaling was investigated in DAMGO-stimulated [(35)S]GTPγS functional assays. Ketamine treatment significantly decreased the bladder volume and isolation decreased the receptor density in cortical membranes. Among all groups, the only change in binding affinity was an increase induced by social isolation in the cortex. G-protein signaling was significantly decreased by either ketamine or social isolation in this tissue. Ketamine treatment, but not housing, significantly increased μ-opioid receptor densities in hippocampal membranes. Both ketamine and isolation increased the efficacy, while the potency of signaling was decreased by any treatment. Ketamine increased the receptor density and G-protein activation; while isolation decreased the efficacy of G-protein signaling in hippocampal membranes. The changes in the co-treated group were similar to those of the isolated animals in most tests. The distinct changes of opioid receptor functioning in different areas of the CNS may, at least partially, explain the augmented nociceptive threshold and morphine potency observed in these animals. Changes in the relative urinary bladder suggest a detrusor hyperreflexia, another sign of schizophrenia. Copyright © 2011 Elsevier B.V. All rights reserved.

Epiretinal membrane negative staining and double peeling in a single block with Brilliant Blue G.

PubMed

Martins, David; Neves, Pedro

2018-01-01

To describe a surgical technique for combined peeling of epiretinal and internal limiting membranes. The authors present their procedure of choice for epiretinal membrane surgery: negative staining effect using Brilliant Blue G and single block removal of the epiretinal and internal limiting membranes in a single step. A total of 26 eyes were operated with the described technique. In all cases, the peeling was performed successfully and with no complications. Minimum postoperative follow-up was 12 months. There were no recurrences of epiretinal membranes. The ideal surgical approach for epiretinal membranes should attempt to reduce mechanical trauma, light exposure, and dye toxicity.

Fabrication and Water Treatment Application of Carbon Nanotubes (CNTs)-Based Composite Membranes: A Review

PubMed Central

Ma, Lining; Dong, Xinfa; Chen, Mingliang; Zhu, Li; Wang, Chaoxian; Yang, Fenglin; Dong, Yingchao

2017-01-01

Membrane separation technology is widely explored for various applications, such as water desalination and wastewater treatment, which can alleviate the global issue of fresh water scarcity. Specifically, carbon nanotubes (CNTs)-based composite membranes are increasingly of interest due to the combined merits of CNTs and membrane separation, offering enhanced membrane properties. This article first briefly discusses fabrication and growth mechanisms, characterization and functionalization techniques of CNTs, and then reviews the fabrication methods for CNTs-based composite membranes in detail. The applications of CNTs-based composite membranes in water treatment are comprehensively reviewed, including seawater or brine desalination, oil-water separation, removal of heavy metal ions and emerging pollutants as well as membrane separation coupled with assistant techniques. Furthermore, the future direction and perspective for CNTs-based composite membranes are also briefly outlined. PMID:28335452

Fabrication and Water Treatment Application of Carbon Nanotubes (CNTs)-Based Composite Membranes: A Review.

PubMed

Ma, Lining; Dong, Xinfa; Chen, Mingliang; Zhu, Li; Wang, Chaoxian; Yang, Fenglin; Dong, Yingchao

2017-03-18

Membrane separation technology is widely explored for various applications, such as water desalination and wastewater treatment, which can alleviate the global issue of fresh water scarcity. Specifically, carbon nanotubes (CNTs)-based composite membranes are increasingly of interest due to the combined merits of CNTs and membrane separation, offering enhanced membrane properties. This article first briefly discusses fabrication and growth mechanisms, characterization and functionalization techniques of CNTs, and then reviews the fabrication methods for CNTs-based composite membranes in detail. The applications of CNTs-based composite membranes in water treatment are comprehensively reviewed, including seawater or brine desalination, oil-water separation, removal of heavy metal ions and emerging pollutants as well as membrane separation coupled with assistant techniques. Furthermore, the future direction and perspective for CNTs-based composite membranes are also briefly outlined.

Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding.

PubMed

Kannan, R; Labotka, R; Low, P S

1988-09-25

Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.

Protection of radiation induced DNA and membrane damages by total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst.

PubMed

Smina, T P; Maurya, D K; Devasagayam, T P A; Janardhanan, K K

2015-05-25

The total triterpenes isolated from the fruiting bodies of Ganoderma lucidum was examined for its potential to prevent γ-radiation induced membrane damage in rat liver mitochondria and microsomes. The effects of total triterpenes on γ-radiation-induced DNA strand breaks in pBR 322 plasmid DNA in vitro and human peripheral blood lymphocytes ex vivo were evaluated. The protective effect of total triterpenes against γ-radiation-induced micronuclei formations in mice bone marrow cells in vivo were also evaluated. The results indicated the significant effectiveness of Ganoderma triterpenes in protecting the DNA and membrane damages consequent to the hazardous effects of radiation. The findings suggest the potential use of Ganoderma triterpenes in radio therapy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Economic evaluation and randomised controlled trial of extracorporeal membrane oxygenation: UK collaborative trial

PubMed Central

Roberts, Tracy E

1998-01-01

Objective: To compare the resource implications and short term outcomes of extracorporeal membrane oxygenation and conventional management for term babies with severe respiratory failure. Design: Cost effectiveness evaluation alongside a randomised controlled trial. Setting: 55 approved recruiting hospitals in the United Kingdom. These hospitals provided conventional management, but infants randomised to extracorporeal membrane oxygenation were transferred to one of five specialist centres. Subjects: 185 mature newborn infants (gestational age at birth >35 weeks, birth weight >2 kg) with severe respiratory failure (oxygenation index >40) recruited between 1993 and 1995. The commonest diagnoses were persistent pulmonary hypertension due to meconium aspiration, congenital diaphragmatic hernia, isolated persistent fetal circulation, sepsis, and idiopathic respiratory distress syndrome. Main outcome measure: Cost effectiveness based on survival at 1 year of age without severe disability. Results: 63 (68%) of the 93 infants randomised to extracorporeal membrane oxygenation survived to 1 year compared with 38 (41%) of the 92 infants who received conventional management. Of those that survived, one infant in each arm was lost to follow up and the proportion with disability at 1 year was similar in the two arms of the trial. One child in each arm had severe disability. The estimated additional cost of extracorporeal membrane oxygenation per additional surviving infant without severe disability was £51 222 and the cost per surviving infant with no disability was £75 327. Conclusions: Extracorporeal membrane oxygenation for term neonates with severe respiratory failure would increase overall survival without disability. Although the policy will increase costs of neonatal health care, it is likely to be as cost effective as other life extending technologies. Key messagesExtracorporeal membrane oxygenation increases survival for term neonates in respiratory failureThe technique was three times more costly than conventional managementIf extracorporeal membrane oxygenation is adopted it will increase the cost of neonatal health care.Extracorporeal membrane oxygenation may be as cost effective as other life extending technologies, but long term follow up studies are needed to confirm this PMID:9756807

Growth of single crystals from solutions using semi-permeable membranes

NASA Astrophysics Data System (ADS)

Varkey, A. J.; Okeke, C. E.

1983-05-01

A technique suitable for growth of single crystals from solutions using semi-preamble membranes is described. Using this technique single crystals of copper sulphate, potassium bromide and ammonium dihydrogen phosphate have been successfully grown. Advantages of this technique over other methods are discussed.

Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

PubMed

Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

2014-08-01

With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells. Copyright © 2014 Elsevier B.V. All rights reserved.

SSM-based electrophysiology.

PubMed

Schulz, Patrick; Garcia-Celma, Juan J; Fendler, Klaus

2008-10-01

An assay technique for the electrical characterization of electrogenic transport proteins on solid supported membranes is presented. Membrane vesicles, proteoliposomes or membrane fragments containing the transporter are adsorbed to the solid supported membrane and are activated by providing a substrate or a ligand via a rapid solution exchange. This technique opens up new possibilities where conventional electrophysiology fails like transporters or ion channels from bacteria and from intracellular compartments. Its rugged design and potential for automation make it suitable for drug screening.

Battacin (Octapeptin B5), a New Cyclic Lipopeptide Antibiotic from Paenibacillus tianmuensis Active against Multidrug-Resistant Gram-Negative Bacteria

PubMed Central

Qian, Chao-Dong; Teng, Yi; Zhao, Wen-Peng; Li, Ou; Fang, Sheng-Guo; Huang, Zhao-Hui; Gao, Hai-Chun

2012-01-01

Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 μg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria. PMID:22183171

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents.

PubMed

Rabani, Vahideh; Davani, Siamak; Gambert-Nicot, Ségolène; Meneveau, Nicolas; Montange, Damien

2016-11-01

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Spectroscopic determination of anthraquinone in kraft pulping liquors using a membrane interface

Treesearch

X.S. Chai; X.T. Yang; Q.X. Hou; J.Y. Zhu; L.-G. Danielsson

2003-01-01

A spectroscopic technique for determining AQ in pulping liquor was developed to effectively separate AQ from dissolved lignin. This technique is based on a flow analysis system with a Nafion membrane interface. The AQ passed through the membrane is converted into its reduced form, AHQ, using sodium hydrosulfite. AHQ has distinguished absorption characteristics in the...

A viable circulating tumor cell isolation device with high retrieval efficiency using a reversibly deformable membrane barrier

NASA Astrophysics Data System (ADS)

Kim, Yoonji; Bu, Jiyoon; Cho, Young-Ho; Son, Il Tae; Kang, Sung-Bum

2017-02-01

Circulating tumor cells (CTCs) contain prognostic information of the tumor, since they shed from the primary tumor and invade into the bloodstream. Therefore, the viable isolation is necessary for a consequent analysis of CTCs. Here, we present a device for the viable isolation and efficient retrieval of CTCs using slanted slot filters, formed by a reversibly deformable membrane barrier. Conventional filters have difficulties in retrieving captured cells, since they easily clog the slots. Moreover, large stress concentration at the sharp edges of squared slots, causes cell lysis. In contrast, the present device shows over 94% of high retrieval efficiency, since the slots can be opened simply by relieving the pressure. Furthermore, the inflated membrane barrier naturally forms the slanted slots, thus reducing the cell damage. By using cancer cell lines, we verified that the present device successfully isolate targeted cells, even at an extremely low concentrations (~10 cells/0.1 ml). In the clinical study, 85.7% of patients initially showed CTC positive while the numbers generally decreased after the surgery. We have also proved that the number of CTCs were highly correlated with tumour invasiveness. Therefore, the present device has potential for use in cancer diagnosis, surgical validation, and invasiveness analysis.

The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles.

PubMed

Antonenkov, Vasily D; Sormunen, Raija T; Hiltunen, J Kalervo

2004-12-01

It has been known for a long time that mammalian peroxisomes are extremely fragile in vitro. Changes in the morphological appearance and leakage of proteins from purified particles demonstrate that peroxisomes are damaged during isolation. However, some properties of purified peroxisomes, e.g., the latency of catalase, imply that their membranes are not disrupted. In the current study, we tried to ascertain the mechanism of this unusual behavior of peroxisomes in vitro. Biochemical and morphological examination of isolated peroxisomes subjected to sonication or to freezing and thawing showed that the membrane of the particles seals after disruption, restoring permeability properties. Transient damage of the membrane leads to the formation of peroxisomal "ghosts" containing nucleoid but nearly devoid of matrix proteins. The rate of leakage of matrix proteins from broken particles depended inversely on their molecular size. The effect of polyethylene glycols on peroxisomal integrity indicated that these particles are osmotically sensitive. Peroxisomes suffered an osmotic lysis during isolation that was resistant to commonly used low-molecular-mass osmoprotectors, e.g., sucrose. Damage to peroxisomes was partially prevented by applying more "bulky" osmoprotectors, e.g., polyethylene glycol 1500. A method was developed for the isolation of highly purified and nearly intact peroxisomes from rat liver by using polyethylene glycol 1500 as an osmoprotector.

Spermatozoa isolated from cat testes retain their structural integrity as well as a developmental potential after refrigeration for up to 7 days.

PubMed

Buarpung, Sirirak; Tharasanit, Theerawat; Thongkittidilok, Chommanart; Comizzoli, Pierre; Techakumphu, Mongkol

2015-10-01

The objective of this study was to compare the efficiency of preservation media for isolated feline testicular spermatozoa as well as the concentrations of bovine serum albumin (BSA) on: (1) the membrane (sperm membrane integrity (SMI)) and DNA integrity of spermatozoa; and (2) the developmental potential of spermatozoa after intracytoplasmic sperm injection (ICSI). Isolated cat spermatozoa were stored in HEPES-M199 medium (HM) or Dulbecco's phosphate-buffered saline (DPBS) at 4°C for up to 7 days. Results indicated that HM maintained a better SMI than DPBS throughout the storage periods (P > 0.05). When spermatozoa were stored in HM supplemented with BSA at different concentrations (4, 8 or 16 mg/ml), SMI obtained from HM containing 8 and 16 mg/ml BSA was higher than with 4 mg/ml BSA (P 0.05). In summary, cat spermatozoa immediately isolated from testicular tissue can be stored as a suspension in basic buffered medium at 4°C for up to 7 days. BSA supplementation into the medium improves membrane integrity of the spermatozoa during cold storage. Testicular spermatozoa stored in HM containing 16 mg/ml BSA retained full in vitro developmental potential after ICSI, similar to that of fresh controls even though DNA integrity had slightly declined.

Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4.

PubMed

Casillo, Angela; Parrilli, Ermenegilda; Filomena, Sannino; Lindner, Buko; Lanzetta, Rosa; Parrilli, Michelangelo; Tutino, Maria Luisa; Corsaro, Maria Michela

2015-07-22

Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by (1)H and (13)C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid.

From micelles to bicelles: Effect of the membrane on particulate methane monooxygenase activity.

PubMed

Ro, Soo Y; Ross, Matthew O; Deng, Yue Wen; Batelu, Sharon; Lawton, Thomas J; Hurley, Joseph D; Stemmler, Timothy L; Hoffman, Brian M; Rosenzweig, Amy C

2018-05-08

Particulate methane monooxygenase (pMMO) is a copper-dependent, integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus ( Mcc. ) capsulatus (Bath) and Methylomicrobium ( Mm. ) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure as observed by electron paramagnetic resonance (EPR) spectroscopy.. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z revealed a mononuclear copper center in the PmoB subunit and indicated that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment, and provide valuable insight into pMMO function. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes[C][W

PubMed Central

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I.

2013-01-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation. PMID:23898030

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

PubMed

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I

2013-06-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

Equilibrium potential for the postsynaptic response in the squid giant synapse.

PubMed

Llinás, R; Joyner, R W; Nicholson, C

1974-11-01

The reversal potential for the EPSP in the squid giant synapse has been studied by means of an intracellular, double oil gap technique. This method allows the electrical isolation of a portion of the axon from the rest of the fiber and generates a quasi-isopotential segment. In order to make the input resistance of this nerve segment as constant as possible, the electroresponsive properties of the nerve membrane were blocked by intracellular injection of tetraethylammonium (TEA) and local extracellular application of tetrodotoxin (TTX). Thus, EPSP's could be evoked in the isolated segment with a minimal amount of electroresponsive properties. The reversal potential for the EPSP (EEPSP) was measured by recording the synaptic potential or the synaptic current during voltage clamping. The results indicate that EEPSP may vary from +15 to +25 mV, which is more positive than would be expected for a 1:1 conductance change for Na(+) and K(+) (approximately -15 mV) and too negative for a pure Na(+) conductance ((+)40 mV). This latter value (E(Na)) was directly determined in the voltage clamp experiments. The results suggest that the synaptic potential is probably produced by a permeability change to Na(+) to K(+) in a 4:1 ratio. No change in time-course was observed in the synaptic current at clamp levels of -100 and +90 mV. The implications of a variable ratio for Na(+)-K(+) permeability in subsynaptic-postsynaptic membranes are discussed.

Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells.

PubMed

Bajenova, O V; Zimmer, R; Stolper, E; Salisbury-Rowswell, J; Nanji, A; Thomas, P

2001-08-17

Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein (hnRNP) M4 gene. The rat ortholog cDNA sequence has not been previously described. The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail. The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript. The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions. When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds (125)I-labeled CEA in a Ca(2+)-dependent fashion. Transfection of rat hnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding. These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells.

Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis.

PubMed

Salerno, Simona; Messina, Antonietta; Giordano, Francesca; Bader, Augustinus; Drioli, Enrico; De Bartolo, Loredana

2017-02-01

Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT-PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications. Copyright © 2016 Elsevier B.V. All rights reserved.

p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

PubMed Central

Yaroslavskiy, B. B.; Stolz, D. B.; Watkins, S. C.; Alber, S. M.; Bradbury, N. A.; Steinman, R. A.

2001-01-01

BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling. PMID:11474127

A simple technique for transferring excised patches of membrane to different solutions for single channel measurements.

PubMed

Quartararo, N; Barry, P H

1987-12-01

A technical problem associated with the patch clamp technique has been the changing of solutions bathing the membrane patch. The simple technique described here solves this problem by means of a movable polythene sleeve placed on the shaft of the patch clamp pipette. The sleeve is initially placed so that the tip of the pipette is exposed. A gigaohm seal is formed using standard techniques. The patch is then excised and the sleeve is slipped down a few mm past the end of the tip of the pipette. When the pipette and sleeve is now removed from the solution, a small drop of solution covering the membrane patch is held in place at the end of the sleeve by surface tension. The pipette is then easily transferred to a different solution without passing the membrane patch through the air-water interface. The sleeve is then simply pulled back up the pipette shaft to expose the membrane patch to the new solution.

Multiple inert gas elimination technique by micropore membrane inlet mass spectrometry--a comparison with reference gas chromatography.

PubMed

Kretzschmar, Moritz; Schilling, Thomas; Vogt, Andreas; Rothen, Hans Ulrich; Borges, João Batista; Hachenberg, Thomas; Larsson, Anders; Baumgardner, James E; Hedenstierna, Göran

2013-10-15

The mismatching of alveolar ventilation and perfusion (VA/Q) is the major determinant of impaired gas exchange. The gold standard for measuring VA/Q distributions is based on measurements of the elimination and retention of infused inert gases. Conventional multiple inert gas elimination technique (MIGET) uses gas chromatography (GC) to measure the inert gas partial pressures, which requires tonometry of blood samples with a gas that can then be injected into the chromatograph. The method is laborious and requires meticulous care. A new technique based on micropore membrane inlet mass spectrometry (MMIMS) facilitates the handling of blood and gas samples and provides nearly real-time analysis. In this study we compared MIGET by GC and MMIMS in 10 piglets: 1) 3 with healthy lungs; 2) 4 with oleic acid injury; and 3) 3 with isolated left lower lobe ventilation. The different protocols ensured a large range of normal and abnormal VA/Q distributions. Eight inert gases (SF6, krypton, ethane, cyclopropane, desflurane, enflurane, diethyl ether, and acetone) were infused; six of these gases were measured with MMIMS, and six were measured with GC. We found close agreement of retention and excretion of the gases and the constructed VA/Q distributions between GC and MMIMS, and predicted PaO2 from both methods compared well with measured PaO2. VA/Q by GC produced more widely dispersed modes than MMIMS, explained in part by differences in the algorithms used to calculate VA/Q distributions. In conclusion, MMIMS enables faster measurement of VA/Q, is less demanding than GC, and produces comparable results.

Nerve membrane ion channels as the target site of environmental toxicants

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

Narahashi, T.

1987-04-01

There are many environmentally important chemicals which exhibit potent effects on the nervous system. Since nerve excitation takes place in a fraction of a second, electrophysiological methods provide the authors with the most straightforward approach to the study of the mechanisms of action of environmental toxicants on the nervous system. Aquatic animals such as crayfish, lobster, squid, and marine snails represent extremely useful materials for such electrophysiological studies, because much of the authors knowledge of nerve excitation is derived from those animals. Nerve excitation takes place as a result of opening and closing of ion channels of the membrane. Thesemore » functions are independent of metabolic energy, and can be measured most effectively by voltage clamp techniques as applied to the giant axons of the crayfish and the squid. Patch clamp techniques developed during the past 10 years have added a new dimension to the electrophysiological investigation. These techniques allow them to measure the activity of individual ion channels, thereby making it possible to analyze the interaction of toxic molecules directly with single ion channels. Examples are given summarizing electrophysiological studies of environmental neurotoxicants. The abdominal nerve cords and neuromuscular preparations isolated from the crayfish are convenient materials for bioassay of certain environmental toxicants such as pyrethroids, chlorinated hydrocarbons, and other insecticides. Only a small fraction of the flux through the sodium channel, less than 1%, must be modified by pyrethroids for the animal to develop symptoms of poisoning. Such a toxicological application from channel to animal is important is understanding the potent toxic effect.« less

Amniotic Membrane Transplantation in Reconstructive and Regenerative Ophthalmology.

PubMed

Röck, Tobias; Bartz-Schmidt, Karl Ulrich; Landenberger, Johanna; Bramkamp, Matthias; Röck, Daniel

2018-03-06

BACKGROUND The goal of this study was to investigate numbers, indications, surgical techniques, and experiences of amniotic membrane transplantation at the University Eye Hospital Tübingen over the last 16 years. MATERIAL AND METHODS Data from all amniotic membrane transplantations from January 2001 to December 2016 were retrospectively analyzed. Data was accessed from the electronic database and the annual reports of the Eye Bank at the University Eye Hospital Tübingen. RESULTS A total of 771 amniotic membrane transplantations were performed between 2001 and 2016 at the University Eye Hospital Tübingen. The mean number of amniotic membrane transplantations was 48 per year (range: 7-81). Overall, the mean number of amniotic membrane transplantations more than doubled, from 31 amniotic membrane transplantations per year during the first 8-year period to 66 amniotic membrane transplantations per year during the second 8-year period (p<0.0001). The most common surgical indications for amniotic membrane transplantation were corneal ulcers and persistent corneal epithelial defects. The inlay, overlay, and sandwich technique became the favored surgical methods for various disorders of the ocular surface. CONCLUSIONS Our study showed a significant increase of amniotic membrane transplantations from 2001 to 2016. This increase is likely influenced by the introduction of different surgical amniotic membrane transplantation techniques, the rising knowledge about containing growth factors, neurotrophins and cytokines, and the demographic change with aging of the population.

Characteristics and functionality of appetite-reducing thylakoid powders produced by three different drying processes.

PubMed

Östbring, Karolina; Sjöholm, Ingegerd; Sörenson, Henrietta; Ekholm, Andrej; Erlanson-Albertsson, Charlotte; Rayner, Marilyn

2018-03-01

Thylakoids, a chloroplast membrane extracted from green leaves, are a promising functional ingredient with appetite-reducing properties via their lipase-inhibiting effect. Thylakoids in powder form have been evaluated in animal and human models, but no comprehensive study has been conducted on powder characteristics. The aim was to investigate the effects of different isolation methods and drying techniques (drum-drying, spray-drying, freeze-drying) on thylakoids' physicochemical and functional properties. Freeze-drying yielded thylakoid powders with the highest lipase-inhibiting capacity. We hypothesize that the specific macromolecular structures involved in lipase inhibition were degraded to different degrees by exposure to heat during spray-drying and drum-drying. We identified lightness (Hunter's L-value), greenness (Hunter's a-value), chlorophyll content and emulsifying capacity to be correlated to lipase-inhibiting capacity. Thus, to optimize the thylakoids functional properties, the internal membrane structure indicated by retained green colour should be preserved. This opens possibilities to use chlorophyll content as a marker for thylakoid functionality in screening processes during process optimization. Thylakoids are heat sensitive, and a mild drying technique should be used in industrial production. Strong links between physicochemical parameters and lipase inhibition capacity were found that can be used to predict functionality. The approach from this study can be applied towards production of standardized high-quality functional food ingredients. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

PubMed

Smith, Adam W

2015-01-01

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy.

PubMed

Albanese, Pascal; Nield, Jon; Tabares, Jose Alejandro Muñoz; Chiodoni, Angelica; Manfredi, Marcello; Gosetti, Fabio; Marengo, Emilio; Saracco, Guido; Barber, James; Pagliano, Cristina

2016-12-01

In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-D-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12-39), as (C 2 S 2 ) 4 and (C 2 S 2  + C 2 S 2 M 2 ) 2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.

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

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.

Antioxidant and membrane effects of procyanidin dimers and trimers isolated from peanut and cocoa.

PubMed

Verstraeten, Sandra V; Hammerstone, John F; Keen, Carl L; Fraga, César G; Oteiza, Patricia I

2005-06-15

The antioxidant and membrane effects of dimer (Dim) and trimer (Trim) procyanidins isolated from cocoa (Theobroma cacao) (B- and C-bonded) and peanut (Arachis hypogea L.) skin (A-bonded) were evaluated in phosphatidyl choline liposomes. When liposomes were oxidized with a steady source of oxidants, the above dimers and trimers inhibited to a similar extent lipid oxidation in a concentration (0.33-5 microM)-dependent manner. With respect to membrane effects, Dim A1, Dim B, Trim A, and Trim C increased (Dim A1 = Dim B and Trim A = Trim C), while Dim A2 decreased, membrane surface potential. All of the procyanidins tested decreased membrane fluidity as determined by fluorescent probes at the water-lipid interface, an effect that extended into the hydrophobic region of the bilayer. Both dimers and trimers protected the lipid bilayer from disruption by Triton X-100. The magnitude of the protection was Dim A1 > Dim A2 > Dim B and Trim C > Trim A. Thus, dimers and trimers can interact with membrane phospholipids, presumably with their polar headgroup. As a consequence of this interaction, they can provide protection against the attack of oxidants and other molecules that challenge the integrity of the bilayer.

Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.

PubMed

Alexandre, Bruno M; Charro, Nuno; Blonder, Josip; Lopes, Carlos; Azevedo, Pilar; Bugalho de Almeida, António; Chan, King C; Prieto, DaRue A; Issaq, Haleem; Veenstra, Timothy D; Penque, Deborah

2012-12-05

Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of Polysulfone Membranes Prepared with Thermally Induced Phase Separation Technique

NASA Astrophysics Data System (ADS)

Tiron, L. G.; Pintilie, Ș C.; Vlad, M.; Birsan, I. G.; Baltă, Ș

2017-06-01

Abstract Membrane technology is one of the most used water treatment technology because of its high removal efficiency and cost effectiveness. Preparation techniques for polymer membranes show an important aspect of membrane properties. Generally, polysulfone (PSf) and polyethersulfone (PES) are used for the preparation of ultrafiltration (UF) membranes. Polysulfone (PSf) membranes have been widely used for separation and purification of different solutions because of their excellent chemical and thermal stability. Polymeric membranes were obtained by phase inversion method. The polymer solution introduced in the nonsolvent bath (distilled water) initiate the evaporation of the solvent from the solution, this phenomenon has a strong influence on the transport properties. The effect of the coagulation bath temperature on the membrane properties is of interest for this study. Membranes are characterized by pure water flux, permeability, porosity and retention of methylene blue. The low temperature of coagulation bath improve the membrane’s rejection and its influence was most notable.

Development of chitosan/pluronic F108/polyethersulfone (PES) nanofiltration (NF) membrane for oily wastewater treatment

NASA Astrophysics Data System (ADS)

Hamzah, Norzakiah; Rohani, Rosiah; Hassan, Abdul Rahman; Sharifuddin, Syazrin Syima; Isa, Mohd Hafez Mohd

2018-06-01

This study discusses a new finding for nanofiltration membrane development using phase inversion technique whereby polyethersulfone (PES) polymer was added with surfactant and additive. This research focuses on the development of a membrane that is efficient in treating oily wastewater and reducing membrane's low permeation flux issues. Five PES nanofiltration membranes were synthesized with pluronic F108 surfactant and different amounts of chitosan additive for each formulation. Subsequently, the effect of adding surfactant and additive on membrane performance was studied. Results showed that the membrane with the optimal amount of chitosan gave the highest flux and the rejection of oily wastewater with up to 90%. In addition, Fourier transform-infrared (FTIR) spectroscopy technique was used to characterize and analyse the membrane's properties. Hence, the developed membranes were successfully characterized and proved to be a good treatment for oily wastewater.

Scalable Production Method for Graphene Oxide Water Vapor Separation Membranes

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

Fifield, Leonard S.; Shin, Yongsoon; Liu, Wei

ABSTRACT Membranes for selective water vapor separation were assembled from graphene oxide suspension using techniques compatible with high volume industrial production. The large-diameter graphene oxide flake suspensions were synthesized from graphite materials via relatively efficient chemical oxidation steps with attention paid to maintaining flake size and achieving high graphene oxide concentrations. Graphene oxide membranes produced using scalable casting methods exhibited water vapor flux and water/nitrogen selectivity performance meeting or exceeding that of membranes produced using vacuum-assisted laboratory techniques. (PNNL-SA-117497)

Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell (PAEC) plasma membrane vesicles

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

Bhat, G.B.; Block, E.R.

1990-02-26

Alterations in the physical state and composition of membrane lipids have been shown to interfere with a number of critical cellular and membrane functions including transmembrane transport. The authors have reported that hypoxia has profound effects upon the physical state and lipid composition of the PAEC plasma membrane bilayer and have suggested that this is responsible for increased serotonin uptake by these cells. In order to determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin, they measured serotonin transport activity (1) in plasma membrane vesicles isolated from normoxic (20% O{sub 2}-5% CO{sub 2}) and hypoxicmore » (0% O{sub 2}-5% CO{sub 2}) PAEC and (2) in PAEC plasma membrane vesicles that were exposed directly to normoxia or hypoxia. A 24-h exposure of PAEC to hypoxia resulted in a 40% increase in specific serotonin transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37C, a 31% increase in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of serotonin transport (normoxia = 3.47 {mu}M versus hypoxia = 3.76 {mu}M) but markedly increased the maximal rate of transport (V{sup max}) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). These results indicate that hypoxia increases serotonin transport in PAEC by a direct effect on the plasma membrane leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for serotonin.« less

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

PubMed

Paul, David; Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

2013-10-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

PubMed Central

Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine

2013-01-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses. PMID:23885072

Glucose rapidly decreases plasma membrane GLUT4 content in rat skeletal muscle.

PubMed

Marette, A; Dimitrakoudis, D; Shi, Q; Rodgers, C D; Klip, A; Vranic, M

1999-02-01

We have previously demonstrated that chronic hyperglycemia per se decreases GLUT4 glucose transporter expression and plasma membrane content in mildly streptozotocin- (STZ) diabetic rats (Biochem. J. 284, 341-348, 1992). In the present study, we investigated the effect of an acute rise in glycemia on muscle GLUT4 and GLUT1 protein contents in the plasma membrane, in the absence of insulin elevation. Four experimental groups of rats were analyzed in the postabsorptive state: 1. Control rats. 2. Hyperglycemic STZ-diabetic rats with moderately reduced fasting insulin levels. 3. STZ-diabetic rats made normoglycemic with phlorizin treatment. 4. Phlorizin-treated (normoglycemic) STZ-diabetic rats infused with glucose for 40 min. The uniqueness of the latter model is that glycemia can be rapidly raised without any concomitant increase in plasma insulin levels. Plasma membranes were isolated from hindlimb muscle and GLUT1 and GLUT4 proteins amounts determined by Western blot analysis. As predicted, STZ-diabetes caused a significant decrease in the abundance of GLUT4 in the isolated plasma membranes. Normalization of glycemia for 3 d with phlorizin treatment restored plasma membrane GLUT4 content in muscle of STZ-diabetic rats. A sudden rise in glycemia over a period of 40 min caused the GLUT4 levels in the plasma membrane fraction to decrease to those of nontreated STZ-diabetic rats. In contrast to the GLUT4 transporter, plasma membrane GLUT1 abundance was not changed by the acute glucose challenge. It is concluded that glucose can have regulatory effect by acutely reducing plasma membrane GLUT4 protein contents in rat skeletal muscle. We hypothesize that this glucose-induced downregulation of plasma membrane GLUT4 could represent a protective mechanism against excessive glucose uptake under hyperglycemic conditions accompanied by insulin resistance.

Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

PubMed

Gubar, O S; Rodnichenko, A E; Vasyliev, R G; Zlatska, A V; Zubov, D O

2017-09-01

We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73 + CD90 + CD105 + CD146 + CD166+CD34 - CD45 - HLA-DR - . HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

Removal of precursors and disinfection by-products (DBPs) by membrane filtration from water; a review.

PubMed

Zazouli, Mohammad Ali; Kalankesh, Laleh R

2017-01-01

Disinfection by-products (DBPs) have heterogeneous structures which are suspected carcinogens as a result of reactions between NOMs (Natural Organic Matter) and oxidants/disinfectants such as chlorine. Because of variability in DBPs characteristics, eliminate completely from drinking water by single technique is impossible. The current article reviews removal of the precursors and DBPs by different membrane filtration methods such as Microfiltration (MF), Ultrafiltration (UF), Nanofiltration (NF) and Reverse Osmosis (RO) techniques. Also, we provide an overview of existing and potentially Membrane filtration techniques, highlight their strengths and drawbacks. MF membranes are a suitable alternative to remove suspended solids and colloidal materials. However, NOMs fractions are effectively removed by negatively charged UF membrane. RO can remove both organic and inorganic DBPs and precursors simultaneously. NF can be used to remove compounds from macromolecular size to multivalent ions.

Morphology of one-time coated palladium-alumina composite membrane prepared by sol-gel process and electroless plating technique

NASA Astrophysics Data System (ADS)

Sari, R.; Dewi, R.; Pardi; Hakim, L.; Diana, S.

2018-03-01

Palladium coated porous alumina ceramic membrane tube was obtained using a combination of sol-gel process and electroless plating technique. The thickness, structure and composition of palladium-alumina composite membrane were analyzed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and atomic force microscopy (AFM). Palladium particle size was 6.18 to 7.64 nm. Palladium membrane with thickness of approximately 301.5 to 815.1 nm was formed at the outer surface of the alumina layer. EDX data confirmed the formation of palladium-alumina membrane containing 45% of palladium. From this research it shows the combination of sol-gel process and electroless plating technique with one-time coating can produce a homogeneous and smoother palladium nano layer film on alumina substrate.

Structures and fabrication techniques for solid state electrochemical devices

DOEpatents

Visco, Steven J.; Jacobson, Craig P.; DeJonghe, Lutgard C.

2006-10-10

Low-cost, mechanically strong, highly electronically conductive porous substrates and associated structures for solid-state electrochemical devices, techniques for forming these structures, and devices incorporating the structures provide solid state electrochemical device substrates of novel composition and techniques for forming thin electrode/membrane/electrolyte coatings on the novel or more conventional substrates. In particular, in one aspect the invention provides techniques for co-firing of device substrate (often an electrode) with an electrolyte or membrane layer to form densified electrolyte/membrane films 5 to 20 microns thick. In another aspect, densified electrolyte/membrane films 5 to 20 microns thick may be formed on a pre-sintered substrate by a constrained sintering process. In some cases, the substrate may be a porous metal, alloy, or non-nickel cermet incorporating one or more of the transition metals Cr, Fe and Cu, or alloys thereof.

Sperm Patch-Clamp

PubMed Central

Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

2014-01-01

Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

Using Raman spectroscopy and SERS for in situ studies of rhizosphere bacteria

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

Mohseni, Hooman; Agahi, Massoud H.; Razeghi, Manijeh

Bacteria colonize plant roots to form a symbiotic relationship with the plant and can play in important role in promoting plant growth. Raman spectroscopy is a useful technique to study these bacterial systems and the chemical signals they utilize to interact with the plant. We present a Raman study of Pantoea YR343 that was isolated from the rhizosphere of Populus deltoides (Eastern Cottonwood). Pantoea sp. YR343 produce yellowish carotenoid pigment that play a role in protection against UV radiation, in the anti-oxidative pathways and in membrane fluidity. Raman spectroscopy is used to non-invasively characterize the membrane bound carotenoids. The spectramore » collected from a mutant strain created by knocking out the crtB gene that encodes a phytoene synthase responsible for early stage of carotenoid biosynthesis, lack the carotenoid peaks. Surface Enhanced Raman Spectroscopy is being employed to detect the plant phytoharmone indoleacetic acid that is synthesized by the bacteria. This work describes our recent progress towards utilizing Raman spectroscopy as a label free, non-destructive method of studying plant-bacteria interactions in the rhizosphere.« less

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

PubMed Central

Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

2012-01-01

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

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

PubMed

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

2017-05-15

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

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.

Evidence for a Na/H Antiporter in Membrane Vesicles Isolated from Roots of the Halophyte Atriplex nummularia.

PubMed

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

1988-05-01

The ATP-dependent establishment of a positive membrane potential (measured as S(14)CN(-)-accumulation) in membrane vesicles isolated from the roots of Atriplex nummularia Lindl. was not inhibited by NaMes and KMes at concentrations up to 140 millimolar. On the other hand, the formation of DeltapH (measured as (14)C-methylamine accumulation or quenching of quinacrine fluorescence), was depressed by NaMes concentrations as low as 30 millimolar. Supply of NaMes after the DeltapH had been established brought about partial dissipation within 30 seconds. Extent of dissipation of DeltapH increased with NaMes concentration over the range tested (up to 180 millimolar). The H(+)/Na(+) exchange indicated by these results was not due to the creation of a Na(+) diffusion potential. Formation of DeltapH in these vesicles was stable to NO(3) (-) up to 100 millimolar; further, the dissipating effect of Na(+) supply was apparent on a DeltapH formed in the presence of 30 millimolar NO(3) (-). Additional evidence that the origin of the membrane vesicles observed in this investigation was not the tonoplast and was probably the plasmalemma included the vanadate sensitivity of the establishment of the membrane potential.

Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells.

PubMed

Wang, Xiaobing; Liu, Quanhong; Wang, Zhezhi; Wang, Pan; Hao, Qiao; Li, Chendi

2009-01-01

The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis. Copyright 2009 S. Karger AG, Basel.

Taspine isolated from Radix et Rhizoma Leonticis inhibits proliferation and migration of endothelial cells as well as chicken chorioallantoic membrane neovascularisation.

PubMed

Zhang, Yanmin; He, Langchong; Meng, Liang; Luo, Wenjuan

2008-01-01

The aim of the present study was to investigate an anti-angiogenic effect of taspine isolated from Radix et Rhizoma Leonticsi. Taspine was screened for the first time, using cell membrane chromatography (CMC). The anti-angiogeneic activity of taspine was tested by using the chicken chorioallantoic membrane (CAM) neovascularisation model in vivo and the HUVEC proliferation and migration models in vitro, respectively. The results showed that taspine could inhibit CAM angiogenesis significantly within the concentration range of 0.5-2 mug/egg, proliferation and migration of endothelial cells in a dose-dependent manner. The CAM histomorphology results indicated that taspine could inhibit blood vessels sprouts and proliferation of vascular endothelial cell. These findings suggest that taspine is a promising candidate for use as an angiogenesis inhibitor.

Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique.

PubMed

Ohvo-Rekilä, Henna; Mattjus, Peter

2011-01-01

The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

PubMed Central

d'Azzo, Alessandra

2013-01-01

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein–protein interactions4,15. PMID:23486347

Membrane-Based Technologies in the Pharmaceutical Industry and Continuous Production of Polymer-Coated Crystals/Particles.

PubMed

Chen, Dengyue; Sirkar, Kamalesh K; Jin, Chi; Singh, Dhananjay; Pfeffer, Robert

2017-01-01

Membrane technologies are of increasing importance in a variety of separation and purification applications involving liquid phases and gaseous mixtures. Although the most widely used applications at this time are in water treatment including desalination, there are many applications in chemical, food, healthcare, paper and petrochemical industries. This brief review is concerned with existing and emerging applications of various membrane technologies in the pharmaceutical and biopharmaceutical industry. The goal of this review article is to identify important membrane processes and techniques which are being used or proposed to be used in the pharmaceutical and biopharmaceutical operations. How novel membrane processes can be useful for delivery of crystalline/particulate drugs is also of interest. Membrane separation technologies are extensively used in downstream processes for bio-pharmaceutical separation and purification operations via microfiltration, ultrafiltration and diafiltration. Also the new technique of membrane chromatography allows efficient purification of monoclonal antibodies. Membrane filtration techniques of reverse osmosis and nanofiltration are being combined with bioreactors and advanced oxidation processes to treat wastewaters from pharmaceutical plants. Nanofiltration with organic solvent-stable membranes can implement solvent exchange and catalyst recovery during organic solvent-based drug synthesis of pharmaceutical compounds/intermediates. Membranes in the form of hollow fibers can be conveniently used to implement crystallization of pharmaceutical compounds. The novel crystallization methods of solid hollow fiber cooling crystallizer (SHFCC) and porous hollow fiber anti-solvent crystallization (PHFAC) are being developed to provide efficient methods for continuous production of polymer-coated drug crystals in the area of drug delivery. This brief review provides a general introduction to various applications of membrane technologies in the pharmaceutical/biopharmaceutical industry with special emphasis on novel membrane techniques for pharmaceutical applications. The method of coating a drug particle with a polymer using the SHFCC method is stable and ready for scale-up for operation over an extended period. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

NANOCAVITY SENSOR ARRAY FOR THE ISOLATION, DETECTION AND QUANTITATION OF ENGINEERED NANOPARTICLES

EPA Science Inventory

Linkage between anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

USDA-ARS?s Scientific Manuscript database

Prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective; they are difficult and expensive to isolate and standardize, and thus often impractical for development and implementation in vaccination programs. In contrast, ind...

Removal of I, Rn, Xe and Kr from off gas streams using PTFE membranes

DOEpatents

Siemer, Darryl D.; Lewis, Leroy C.

1990-01-01

A process for removing I, R, Xe and Kr which involves the passage of the off gas stream through a tube-in-shell assembly, whereby the tubing is a PTFE membrane which permits the selective passages of the gases for removing and isolating the gases.

Removal of I, Rn, Xe and Kr from off gas streams using PTFE membranes

DOEpatents

Siemer, Darryl D.; Lewis, Leroy C.

1990-08-07

A process for removing I, R, Xe and Kr which involves the passage of the off gas stream through a tube-in-shell assembly, whereby the tubing is a PTFE membrane which permits the selective passages of the gases for removing and isolating the gases.

Tolerance to chitosan by Trichoderma species is associated with low membrane fluidity.

PubMed

Zavala-González, Ernesto A; Lopez-Moya, Federico; Aranda-Martinez, Almudena; Cruz-Valerio, Mayra; Lopez-Llorca, Luis Vicente; Ramírez-Lepe, Mario

2016-07-01

The effect of chitosan on growth of Trichoderma spp., a cosmopolitan genus widely exploited for their biocontrol properties was evaluated. Based on genotypic (ITS of 18S rDNA) characters, four isolates of Trichoderma were identified as T. pseudokoningii FLM16, T. citrinoviride FLM17, T. harzianum EZG47, and T. koningiopsis VSL185. Chitosan reduces radial growth of Trichoderma isolates in concentration-wise manner. T. koningiopsis VSL185 was the most chitosan tolerant isolate in all culture media amended with chitosan (0.5-2.0 mg ml(-1) ). Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were determined showing that T. koningiopsis VSL185 displays higher chitosan tolerance with MIC value >2000 μg ml(-1) while for other Trichoderma isolates MIC values were around 10 μg ml(-1) . Finally, free fatty acid composition reveals that T. koningiopsis VSL185, chitosan tolerant isolate, displays lower linolenic acid (C18:3) content than chitosan sensitive Trichoderma isolates. Our findings suggest that low membrane fluidity is associated with chitosan tolerance in Trichoderma spp. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas.

PubMed

Valenti, Daniela; de Bari, Lidia; De Filippis, Bianca; Ricceri, Laura; Vacca, Rosa Anna

2014-01-01

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

PubMed Central

Santarius, K A; Giersch, C

1984-01-01

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature. PMID:6478028

Free-standing membrane polymer laser on the end of an optical fiber

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

Zhai, Tianrui, E-mail: trzhai@bjut.edu.cn, E-mail: zhangxinping@bjut.edu.cn; Li, Songtao; Hu, Yujie

2016-01-25

One- and two-dimensional distributed feedback cavities were constructed on free-standing polymer membranes using spin-coating and lift-off techniques. Low threshold lasing was generated through feedback amplification when the 290-nm membrane device was optically pumped, which was attributed to the strong confinement mechanism provided by the active waveguide layer without a substrate. The free-standing membrane polymer laser is flexible and can be transplanted. Single- and dual-wavelength fiber lasers were achieved by directly attaching the membrane polymer laser on the optical fiber end face. This technique provides potential to fabricate polymer lasers on surfaces with arbitrary shapes.

Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification

PubMed Central

Liao, Pin-Chao; Boldogh, Istvan R.; Siegmund, Stephanie E.

2018-01-01

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods. PMID:29698455

Free-Energy Profiles of Membrane Insertion of the M2 Transmembrane Peptide from Influenza A Virus

DTIC Science & Technology

2008-12-01

ABSTRACT The insertion of the M2 transmembrane peptide from influenza A virus into a membrane has been studied with molecular - dynamics simulations ...performed replica-exchange molecular - dynamics simulations with umbrella-sampling techniques to characterize the probability distribution and conformation...atomic- detailed molecular dynamics (MD) simulation techniques represent a valuable complementary methodology to inves- tigate membrane-insertion of

Optimization of hydrothermal pretreatment and membrane filtration processes of various feedstocks to isolate hemicelluloses for biopolymer applications

NASA Astrophysics Data System (ADS)

Sukhbaatar, Badamkhand

Hemicelluloses (HC) are the second most abundant plant polysaccharides after cellulose, constituting 25-30% of plant materials. In spite of their abundance, HC are not effectively utilized. Recently, considerable interest has been directed to HC-based biomaterials because of their high oxygen barrier properties, which has potential in food packaging applications. In this study, HC were extracted from sugarcane bagasse and southern yellow pine using a hydrothermal technique which utilizes hot compressed water without catalyst. The parameters affecting the yield of extracted HC such as temperature, time and pressure, were tested and optimized. Eighty four percent of xylose was extracted from sugarcane bagasse at the optimum condition, 180 °C 30 min and 1 MPa pressure. In the case of southern yellow pine, 79% of the mannose was extracted at 190 °C for 10 min and 2 MPa pressure. Concentration and isolation of HC from bagasse and southern yellow pine HC extract were performed by membrane filtration and freeze drying systems. Isolated HC were characterized by FT-IR and 13C NMR techniques and used as a starting material for film preparation. Films were prepared in 0/100, 50/50, 60/40, 70/30 and 80/20% ratios of HC and sodium carboxymethylcellulose (CMC). Thirty five percent of sorbitol (w/w of HC and CMC weight) was also added as a plasticizer. Films were evaluated by measuring water absorption, water vapor permeability (WVP), tensile property and oxygen barrier capability. At 55% relative humidity (RH) and 25 °C the water absorption of both sugarcane bagasse and southern yellow pine HC-based films tended to increase as HC content increased. The lowest WVP of sugarcane bagasse (3.84e-12 g/Pa h m) and southern yellow pine HC films (2.18e-12 g/Pa h m) were determined in 60/40 HC/CMC films. Tensile test results showed that as HC content increases the Young's modulus decreases, deflection at maximum load and percentage of strain at break increase. It implies that the film properties are changing from stiff to elastic. The oxygen permeability for 60/40 bagasse HC/CMC film was 0.005265 cc μm / (m2 day kPa) and for 70/30 pine HC/CMC film was 0.007570 cc μm / (m2 day kPa).

Structures And Fabrication Techniques For Solid State Electrochemical Devices

DOEpatents

Visco, Steven J.; Jacobson, Craig P.; DeJonghe, Lutgard C.

2005-12-27

Provided are low-cost, mechanically strong, highly electronically conductive porous substrates and associated structures for solid-state electrochemical devices, techniques for forming these structures, and devices incorporating the structures. The invention provides solid state electrochemical device substrates of novel composition and techniques for forming thin electrode/membrane/electrolyte coatings on the novel or more conventional substrates. In particular, in one embodiment the invention provides techniques for co-firing of device substrate (often an electrode) with an electrolyte or membrane layer to form densified electrolyte/membrane films 5 to 20 microns thick. In another embodiment, densified electrolyte/membrane films 5 to 20 microns thick may be formed on a pre-sintered substrate by a constrained sintering process. In some cases, the substrate may be a porous metal, alloy, or non-nickel cermet incorporating one or more of the transition metals Cr, Fe, Cu and Ag, or alloys thereof.

Structures and fabrication techniques for solid state electrochemical devices

DOEpatents

Visco, Steven J.; Jacobson, Craig P.; DeJonghe, Lutgard C.

2003-08-12

Provided are low-cost, mechanically strong, highly electronically conductive porous substrates and associated structures for solid-state electrochemical devices, techniques for forming these structures, and devices incorporating the structures. The invention provides solid state electrochemical device substrates of novel composition and techniques for forming thin electrode/membrane/electrolyte coatings on the novel or more conventional substrates. In particular, in one embodiment the invention provides techniques for co-firing of device substrate (often an electrode) with an electrolyte or membrane layer to form densified electrolyte/membrane films 5 to 20 microns thick. In another embodiment, densified electrolyte/membrane films 5 to 20 microns thick may be formed on a pre-sintered substrate by a constrained sintering process. In some cases, the substrate may be a porous metal, alloy, or non-nickel cermet incorporating one or more of the transition metals Cr, Fe, Cu and Ag, or alloys thereof.

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

PubMed

Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

2009-01-01

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Proteomic approaches and their application to plant gravitropism.

PubMed

Basu, Proma; Luesse, Darron R; Wyatt, Sarah E

2015-01-01

Proteomics is a powerful technique that allows researchers a window into how an organism responds to a mutation, a specific environment, or at a distinct point during development by quantifying relative protein abundance and posttranslational modifications. Here, we describe methods for the proteomic analysis of Arabidopsis thaliana tissue. Extraction protocols are provided for isolation of soluble, plasma membrane, and tonoplast proteins. In addition, basic analysis and quality metrics for MS/MS data are discussed. The protocols outlined have the potential to unlock new avenues of research that are not possible through basic genetics or transcriptomic approaches. By combining proteomic information with known gene regulatory patterns, researchers can gain a complete picture of how molecular pathways, such as those required for gravitropism, are initiated, regulated, and terminated.

The styrene-maleic acid copolymer: a versatile tool in membrane research.

PubMed

Dörr, Jonas M; Scheidelaar, Stefan; Koorengevel, Martijn C; Dominguez, Juan J; Schäfer, Marre; van Walree, Cornelis A; Killian, J Antoinette

2016-01-01

A new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene-maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein-lipid and protein-protein interactions.

Membrane order in the plasma membrane and endocytic recycling compartment.

PubMed

Iaea, David B; Maxfield, Frederick R

2017-01-01

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

Membrane order in the plasma membrane and endocytic recycling compartment

PubMed Central

Iaea, David B.; Maxfield, Frederick R.

2017-01-01

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

The G protein alpha subunit (GP alpha1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower.

PubMed

Weiss, C A; White, E; Huang, H; Ma, H

1997-05-05

Towards the elucidation of the cellular function(s) of GP alpha1, we have characterized its subcellular localization using immunofluorescence and cell fractionation. GP alpha1 is not present in nuclei or chloroplasts. It is a membrane-bound protein, and analysis of isolated endoplasmic and plasma membranes indicates a good correlation between GP alpha1 in both the plasma membrane and the ER compartment. Interestingly, these results may suggest more different functions for GP alpha1: it might be involved in transmission of extracellular signals across the plasma membrane and in the cytoplasm, and/or it may also be involved in regulating some aspects of the ER functions or membrane trafficking between both membranes.

Adhesion between liposomes mediated by the chlorophyll a/b light- harvesting complex isolated from chloroplast membranes

PubMed Central

1980-01-01

A highly purified chlorophyll a/b light-harvesting complex (chl a/b LHC; chl a/b ratio 1.2) was obtained from Triton-solubilized chloroplast membranes of pea and barley according to the method of Burke et al. (1978, Arch. Biochem. Biophys. 187: 252--263). Gel electrophoresis of the cation-precipitated chl a/b LHC from peas reveals the presence of four polypeptides in the 23- to 28-kdalton size range. Three of these peptides appear to be identical to those derived from re-electrophoresed CPII and CPII* bands. In freeze-fracture replicas, the cation-precipitated chl a/b LHC appears as a semicrystalline aggregate of membranous sheets containing closely spaced granules. Upon removal of the cations by dialysis, the aggregates break up into their constituent membranous sheets without changing their granular substructure. These membranous sheets can be resolubilized in 1.5% Triton X-100, and the chl a/b LHC particles then reconstituted into soybean lecithin liposomes. Freeze-fracture micrographs of the reconstituted chl a/b LHC vesicles suspended in a low salt medium reveal randomly dispersed approximately 80-A particles on both concave and convex fracture faces as well as some crystalline particle arrays, presumably resulting from incompletely solubilized fragments of the membranous sheets. Based on the approximately 80-A diameter of the particles, and on the assumption that one freeze- fracture particle represents the structural unit of one chl a/b LHC aggregate, a theoretical mol wt of approximately 200 kdalton has been calculated for the chl a/b LHC. Deep-etching and negative-staining techniques reveal that the chl a/b LHC particles are also exposed on the surface of the bilayer membranes. Addition of greater than or equal to 2 mM MgCl2 or greater than or equal to 60 mM NaCl to the reconstituted vesicles leads to their aggregation and, with divalent cations, to the formation of extensive membrane stacks. At the same time, the chl a/b LHC particles become clustered into the adhering membrane regions. Under these conditions the particles in adjacent membranes usually become precisely aligned. Evidence is presented to aupport the hypothesis that adhesion between the chl a/b LHC particles is mediated by hydrophobic interactions, and that the cations are needed to neutralize surface charges on the particles. PMID:7350170

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels.

PubMed

Brzica, Hrvoje; Abdullahi, Wazir; Reilly, Bianca G; Ronaldson, Patrick T

2018-05-07

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

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

PubMed Central

Xiao, Z; Devreotes, P N

1997-01-01

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

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

PubMed

Xiao, Z; Devreotes, P N

1997-05-01

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

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

PubMed Central

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

1990-01-01

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

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

PubMed

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

1990-12-01

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

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.

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles.

PubMed

Inoue, M; Arias, I M

1988-07-01

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.

[Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide].

PubMed

Kuleshova, L G; Gordienko, E A; Kovalenko, I F

2014-01-01

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

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 via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

Antifouling membranes for sustainable water purification: strategies and mechanisms.

PubMed

Zhang, Runnan; Liu, Yanan; He, Mingrui; Su, Yanlei; Zhao, Xueting; Elimelech, Menachem; Jiang, Zhongyi

2016-10-24

One of the greatest challenges to the sustainability of modern society is an inadequate supply of clean water. Due to its energy-saving and cost-effective features, membrane technology has become an indispensable platform technology for water purification, including seawater and brackish water desalination as well as municipal or industrial wastewater treatment. However, membrane fouling, which arises from the nonspecific interaction between membrane surface and foulants, significantly impedes the efficient application of membrane technology. Preparing antifouling membranes is a fundamental strategy to deal with pervasive fouling problems from a variety of foulants. In recent years, major advancements have been made in membrane preparation techniques and in elucidating the antifouling mechanisms of membrane processes, including ultrafiltration, nanofiltration, reverse osmosis and forward osmosis. This review will first introduce the major foulants and the principal mechanisms of membrane fouling, and then highlight the development, current status and future prospects of antifouling membranes, including antifouling strategies, preparation techniques and practical applications. In particular, the strategies and mechanisms for antifouling membranes, including passive fouling resistance and fouling release, active off-surface and on-surface strategies, will be proposed and discussed extensively.

Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

PubMed Central

Ogunnariwo, J A; Schryvers, A B

1990-01-01

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

Sodium ion conducting polymer electrolyte membrane prepared by phase inversion technique

NASA Astrophysics Data System (ADS)

Harshlata, Mishra, Kuldeep; Rai, D. K.

2018-04-01

A mechanically stable porous polymer membrane of Poly(vinylidene fluoride-hexafluoropropylene) has been prepared by phase inversion technique using steam as a non-solvent. The membrane possesses semicrystalline network with enhanced amorphicity as observed by X-ray diffraction. The membrane has been soaked in an electrolyte solution of 0.5M NaPF6 in Ethylene Carbonate/Propylene Carbonate (1:1) to obtain the gel polymer electrolyte. The porosity and electrolyte uptake of the membrane have been found to be 67% and 220% respectively. The room temperature ionic conductivity of the membrane has been obtained as ˜ 0.3 mS cm-1. The conductivity follows Arrhenius behavior with temperature and gives activation energy as 0.8 eV. The membrane has been found to possess significantly large electrochemical stability window of 5.0 V.

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.

PubMed

Renault, Thibaud T; Luna-Vargas, Mark P A; Chipuk, Jerry E

2016-08-05

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al ., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al ., 2015).

Method of detecting defects in ion exchange membranes of electrochemical cells by chemochromic sensors

DOEpatents

Brooker, Robert Paul; Mohajeri, Nahid

2016-01-05

A method of detecting defects in membranes such as ion exchange membranes of electrochemical cells. The electrochemical cell includes an assembly having an anode side and a cathode side with the ion exchange membrane in between. In a configuration step a chemochromic sensor is placed above the cathode and flow isolation hardware lateral to the ion exchange membrane which prevents a flow of hydrogen (H.sub.2) between the cathode and anode side. The anode side is exposed to a first reactant fluid including hydrogen. The chemochromic sensor is examined after the exposing for a color change. A color change evidences the ion exchange membrane has at least one defect that permits H.sub.2 transmission therethrough.

Formation and characterization of asymmetric polyimide hollow fiber membranes for gas separations

NASA Astrophysics Data System (ADS)

Clausi, Dominic Thomas

Ultra-thin and virtually defect-free polyimide hollow fiber membranes were formed using a "dry/wet" type spinning solution. Fibers were spun from the commercially available polyimide, Matrimidsp{°ler}, using a dry-jet, wet quench spinning apparatus. Spin dopes were comprised of volatile and non-volatile solvents, polymer, and non-solvent. The influence of dope composition, spinning parameters, and dehydration procedures on the membrane morphology and performance was investigated. Without post-treatment, the fibers exhibited skin thicknesses less than 1000 A and Osb2/Nsb2 selectivities within 90% of those determined for dense, solution-cast films. The 250 mum O.D./125 mum I.D. fibers were spun at take-up rates comparable to those used in commercial processes and had macrovoid-free morphologies. A new characterization technique has also been developed where a permeating gas is held at constant transmembrane pressure while the average pressure in the porous support of an asymmetric membrane is varied. This alters the mean free path of gas molecules permeating through the substructure while maintaining a constant driving force for permeation. This technique characterizes the magnitude of the substructure resistance and its pressure dependence, thereby providing a means to compare the morphologies of different membrane samples. Well defined composite-laminate membranes were constructed to validate this technique, which was subsequently used to characterize the substructures of the hollow fiber membranes formed in this work. Two additional rapid characterization techniques have been developed for use before fiber dehydration (i.e., wet fibers). These techniques probe the membrane skin layer with aqueous solutions of disperse dyes and poly(ethylene glycol), respectively. Fiber skin integrity can be characterized using these techniques prior to lengthy downstream processing (i.e., solvent exchange, drying, and post-treatment), providing quick elucidation of membrane skin morphology. Finally, a qualitative model describing the skin layer morphology of phase inversion membranes has been developed. This model arose from observed differences in the permeation characteristics of highly sorbing gases between bore and shell side feeds. It is proposed that the skin layer contains an asymmetric distribution of unrelaxed volume introduced during the formation process. This model has been successfully tested with COsb2/CHsb4 permeation measurements conducted at varying temperatures and feed configurations.

Characterization of PSII-LHCII supercomplexes isolated from pea thylakoid membrane by one-step treatment with α- and β-dodecyl-D-maltoside.

PubMed

Barera, Simone; Pagliano, Cristina; Pape, Tillmann; Saracco, Guido; Barber, James

2012-12-19

It was the work of Jan Anderson, together with Keith Boardman, that showed it was possible to physically separate photosystem I (PSI) from photosystem II (PSII), and it was Jan Anderson who realized the importance of this work in terms of the fluid-mosaic model as applied to the thylakoid membrane. Since then, there has been a steady progress in the development of biochemical procedures to isolate PSII and PSI both for physical and structural studies. Dodecylmaltoside (DM) has emerged as an effective mild detergent for this purpose. DM is a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric centre of the carbohydrate head group, axial in α-DM and equatorial in β-DM. We have compared the use of α-DM and β-DM for the isolation of supramolecular complexes of PSII by a single-step solubilization of stacked thylakoid membranes isolated from peas. As a result, we have optimized conditions to obtain homogeneous preparations of the C(2)S(2)M(2) and C(2)S(2) supercomplexes following the nomenclature of Dekker & Boekema (2005 Biochim. Biophys. Acta 1706, 12-39). These PSII-LHCII supercomplexes were subjected to biochemical and structural analyses.

Low K+-induced hyperpolarizations trigger transient depolarizations and action potentials in rabbit ventricular myocytes

PubMed Central

Akuzawa-Tateyama, M; Tateyama, M; Ochi, R

1998-01-01

The effects of large reductions of [K+]o on membrane potential were studied in isolated rabbit ventricular myocytes using the whole-cell patch clamp technique.Decreasing [K+]o from the normal level of 5.4 mm to 0.1 mm increased resting membrane potential (Vrest) from −75.6 ± 0.3 to −140.3 ± 1.9 mV (means ± s.e.m; n = 127), induced irregular, transient depolarizations with mean maximal amplitudes of 19.5 ± 1.5 mV and elicited action potentials in 56.7 % of trials. The action potentials exhibited overshoots of 37.9 ± 1.5 mV (n = 72) and sustained plateaux.Addition of 0.1 mm La3+ in the presence of 0.1 mm[K+]o significantly increased Vrest but decreased the amplitude of transient depolarizations and suppressed the firing of action potentials.Replacement of external Na+ or Cl− with N-methyl-D-glucamine or aspartate, respectively, or internal dialysis with 10 mm EGTA or BAPTA had little effect on low [K+]o-induced membrane potential changes.Hyperpolarizing voltage clamp pulses to potentials between −110 and −200 mV activated irregular inward currents that increased in amplitude and frequency with increasing hyperpolarization and were depressed by 0.1 mm La3+.The generation of transient depolarizations by low [K+]o can be explained as being a consequence of decreasing the inward rectifier K+ current (IK1) and the appearance of inward currents reflecting electroporation resulting from strong electric fields across the membrane. PMID:9824717

A new oil/membrane approach for integrated sweat sampling and sensing: sample volumes reduced from μL's to nL's and reduction of analyte contamination from skin.

PubMed

Peng, R; Sonner, Z; Hauke, A; Wilder, E; Kasting, J; Gaillard, T; Swaille, D; Sherman, F; Mao, X; Hagen, J; Murdock, R; Heikenfeld, J

2016-11-01

Wearable sweat biosensensing technology has dominantly relied on techniques which place planar-sensors or fluid-capture materials directly onto the skin surface. This 'on-skin' approach can result in sample volumes in the μL regime, due to the roughness of skin and/or due to the presence of hair. Not only does this increase the required sampling time to 10's of minutes or more, but it also increases the time that sweat spends on skin and therefore increases the amount of analyte contamination coming from the skin surface. Reported here is a first demonstration of a new paradigm in sweat sampling and sensing, where sample volumes are reduced from the μL's to nL's regime, and where analyte contamination from skin is reduced or even eliminated. A micro-porous membrane is constructed such that it is porous to sweat only. To complete a working device, first placed onto skin is a cosmetic-grade oil, secondly this membrane, and thirdly the sensors. As a result, spreading of sweat is isolated to only regions above the sweat glands before it reaches the sensors. Best case sampling intervals are on the order of several minutes, and the majority of hydrophilic (low oil solubility) contaminants from the skin surface are blocked. In vitro validation of this new approach is performed with an improved artificial skin including human hair. In vivo tests show strikingly consistent results, and reveal that the oil/membrane is robust enough to even allow horizontal sliding of a sensor.

Nanoscale Membrane Curvature detected by Polarized Localization Microscopy

NASA Astrophysics Data System (ADS)

Kelly, Christopher; Maarouf, Abir; Woodward, Xinxin

Nanoscale membrane curvature is a necessary component of countless cellular processes. Here we present Polarized Localization Microscopy (PLM), a super-resolution optical imaging technique that enables the detection of nanoscale membrane curvature with order-of-magnitude improvements over comparable optical techniques. PLM combines the advantages of polarized total internal reflection fluorescence microscopy and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without reducing localization precision by point spread function manipulation. PLM resolved nanoscale membrane curvature of a supported lipid bilayer draped over polystyrene nanoparticles on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, PLM provides single-molecule trajectories and the aggregation of curvature-inducing proteins with super-resolution to reveal the correlated effects of membrane curvature, dynamics, and molecular sorting. For example, cholera toxin subunit B has been observed to induce nanoscale membrane budding and concentrate at the bud neck. PLM reveals a previously hidden and critical information of membrane topology.

Isolation of Chromobacterium spp. from foods, soil, and water.

PubMed Central

Koburger, J A; May, S O

1982-01-01

Chromobacterium violaceum, a soil and water inhabitant, has been implicated in human disease with a high mortality rate, particularly in the southeastern United States. The psychrotrophic Chromobacterium lividum has been isolated from foods, water, and soil, but is not considered pathogenic. To determine the distribution of Chromobacterium spp. in soil, water, and foods in the Gainesville area, we evaluated Bennett, Ryalls and Moss, and Aeromonas membrane agars for their ability to recover these organisms from various samples when incubated at 25 or 35 degrees C. Bennett agar was best for the isolation of both species when incubated at 25 degrees C; however, at 35 degrees C, Aeromonas membrane agar gave the highest recoveries of C. violaceum. C. violaceum was recovered only from soil and water, whereas C. lividum was frequently recovered from foods as well as soil and water. PMID:7159087

Mechanism and pharmacological rescue of berberine-induced hERG channel deficiency

PubMed Central

Yan, Meng; Zhang, Kaiping; Shi, Yanhui; Feng, Lifang; Lv, Lin; Li, Baoxin

2015-01-01

Berberine (BBR), an isoquinoline alkaloid mainly isolated from plants of Berberidaceae family, is extensively used to treat gastrointestinal infections in clinics. It has been reported that BBR can block human ether-a-go-go-related gene (hERG) potassium channel and inhibit its membrane expression. The hERG channel plays crucial role in cardiac repolarization and is the target of diverse proarrhythmic drugs. Dysfunction of hERG channel can cause long QT syndrome. However, the regulatory mechanisms of BBR effects on hERG at cell membrane level remain unknown. This study was designed to investigate in detail how BBR decreased hERG expression on cell surface and further explore its pharmacological rescue strategies. In this study, BBR decreases caveolin-1 expression in a concentration-dependent manner in human embryonic kidney 293 (HEK293) cells stably expressing hERG channel. Knocking down the basal expression of caveolin-1 alleviates BBR-induced hERG reduction. In addition, we found that aromatic tyrosine (Tyr652) and phenylalanine (Phe656) in S6 domain mediate the long-term effect of BBR on hERG by using mutation techniques. Considering both our previous and present work, we propose that BBR reduces hERG membrane stability with multiple mechanisms. Furthermore, we found that fexofenadine and resveratrol shorten action potential duration prolongated by BBR, thus having the potential effects of alleviating the cardiotoxicity of BBR. PMID:26543354

Investigation of terpinen-4-ol effects on vascular smooth muscle relaxation.

PubMed

Maia-Joca, Rebeca Peres Moreno; Joca, Humberto Cavalcante; Ribeiro, Francisca Jéssica Penha; do Nascimento, Renata Vieira; Silva-Alves, Kerly Shamyra; Cruz, Jader S; Coelho-de-Souza, Andrelina Noronha; Leal-Cardoso, José Henrique

2014-10-12

This study investigated the mechanisms underlying the vascular effects of terpinen-4-ol in isolated rat aortic ring preparations. The thoracic aortae of healthy rats were submitted to isometric tension recording. Membrane resting potential and input membrane resistance were measured by conventional microelectrode technique. Terpinen-4-ol reversibly relaxed endothelium-containing preparations pre-contracted with high K(+) and phenylephrine with IC50 values of 421.43 μM and 802.50 μM, respectively. These effects were significantly reduced by vascular endothelium removal. In Ca(2+)-free and high K(+) (80 mM) medium, the contractions produced by Ba(2+) were reduced by terpinen-4-ol (100-1000 μM) in a concentration-dependent manner. In aortic rings maintained under Ca(2+)-free conditions, terpinen-4-ol significantly reduced the contractions induced by either phenylephrine (1 μM) or phorbol 12,13-dibutyrate (1 μM). Terpinen-4-ol (10-1000 μM) also relaxed the contractions evoked by BAYK-8644 (3 μM) with an IC50 of 454.23 μM. Neither membrane resting potential nor input resistance of smooth muscle cells was altered by terpinen-4-ol exposure. The present results suggest that terpinen-4-ol induced vascular smooth muscle relaxation that was preferentially due to the inhibition of electromechanical pathways related to calcium influx through voltage-operated calcium channels. Copyright © 2014 Elsevier Inc. All rights reserved.

A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

PubMed

Wertheimer, Christian; Eibl-Lindner, Kirsten H; Compera, Denise; Kueres, Alexander; Wolf, Armin; Docheva, Denitsa; Priglinger, Siegfried G; Priglinger, Claudia; Schumann, Ricarda G

2017-11-01

To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

Phospholipid composition of the plasma membrane of the green alga, Hydrodictyon africanum.

PubMed Central

Bailey, D S; Northcote, D H

1976-01-01

A plasma-membrane fraction was isolated from the alga Hydrodictyon africanum by micro-dissection and its phospholipid components were analysed. Phosphatidylcholine was the major phospholipid of the preparation. Both phosphatidylserine and diphosphatidylglycerol were enriched in the fraction compared with the whole cell, but the relative amount of phosphatidylglycerol present was less than that in the whole cell. Phosphatidylinositol was absent from the plasma-membrane preparation. Images PLATE 1 PLATE 2 PMID:182144

Development of an automation technique for the establishment of functional lipid bilayer arrays

NASA Astrophysics Data System (ADS)

Hansen, J. S.; Perry, M.; Vogel, J.; Vissing, T.; Hansen, C. R.; Geschke, O.; Emnéus, J.; Nielsen, C. H.

2009-02-01

In the present work, a technique for establishing multiple black lipid membranes (BLMs) in arrays of micro structured ethylene tetrafluoroethylene (ETFE) films, and supported by a micro porous material was developed. Rectangular 8 × 8 arrays with apertures having diameters of 301 ± 5 µm were fabricated in ETFE Teflon film by laser ablation using a carbon dioxide laser. Multiple lipid membranes could be formed across the micro structured 8 × 8 array ETFE partitions. Success rates for the establishment of cellulose-supported BLMs across the multiple aperture arrays were above 95%. However, the time course of the membrane thinning process was found to vary considerably between multiple aperture bilayer experiments. An airbrush partition pretreatment technique was developed to increase the reproducibility of the multiple lipid bilayers formation during the time course from the establishment of the lipid membranes to the formation of bilayers. The results showed that multiple lipid bilayers could be reproducible formed across the airbrush-pretreated 8 × 8 rectangular arrays. The ionophoric peptide valinomycin was incorporated into established membrane arrays, resulting in ionic currents that could be effectively blocked by tetraethylammonium. This shows that functional bimolecular lipid membranes were established, and furthermore outlines that the established lipid membrane arrays could host functional membrane-spanning molecules.

Mechanism regulating nuclear calcium signaling.

PubMed

Malviya, Anant N; Klein, Christian

2006-01-01

Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca(2+)-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-gamma1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP(3) generating system.

Treatment of table olive processing wastewaters using novel photomodified ultrafiltration membranes as first step for recovering phenolic compounds.

PubMed

Garcia-Ivars, Jorge; Iborra-Clar, Maria-Isabel; Alcaina-Miranda, Maria-Isabel; Mendoza-Roca, José-Antonio; Pastor-Alcañiz, Laura

2015-06-15

Table olive processing wastewaters (TOPW) have high salt concentration and total phenolic content (TPC) causing many environmental problems. To reduce them, ultrafiltration (UF) was applied for treating TOPW. However, NaCl, which is the main responsible of salinity in TOPW, and phenols are small molecules that cannot be separated by conventional UF membranes. They have serious problems caused by fouling, which can be overcome using membrane modification techniques. For these reasons, photomodification may be an effective technique to obtain a stream rich in TPC due to the changes in membrane surface properties. UV-modification in the presence of two hydrophilic compounds (polyethylene glycol and aluminium oxide) was performed to achieve membranes with high reductions of organic matter and to keep the TPC as high as possible. Commercial polyethersulfone (PES) membranes of 30 kDa were used. Surface modification was evaluated using FTIR-ATR spectroscopy and membrane performance was studied by calculating the rejection ratios of colour, chemical oxygen demand (COD) and TPC. Results demonstrated that UF is a useful pre-treatment to reduce organic matter from TOPW, obtaining a permeate rich in TPC. PES/Al2O3 membranes displayed superior antifouling properties and rejection values, keeping high the TPC (>95%). Therefore, UF using modified membranes is an appropriate and sustainable technique for treating TOPW. Copyright © 2015 Elsevier B.V. All rights reserved.

Diffusive Silicon Nanopore Membranes for Hemodialysis Applications

PubMed Central

Kim, Steven; Feinberg, Benjamin; Kant, Rishi; Chui, Benjamin; Goldman, Ken; Park, Jaehyun; Moses, Willieford; Blaha, Charles; Iqbal, Zohora; Chow, Clarence; Wright, Nathan; Fissell, William H.; Zydney, Andrew; Roy, Shuvo

2016-01-01

Hemodialysis using hollow-fiber membranes provides life-sustaining treatment for nearly 2 million patients worldwide with end stage renal disease (ESRD). However, patients on hemodialysis have worse long-term outcomes compared to kidney transplant or other chronic illnesses. Additionally, the underlying membrane technology of polymer hollow-fiber membranes has not fundamentally changed in over four decades. Therefore, we have proposed a fundamentally different approach using microelectromechanical systems (MEMS) fabrication techniques to create thin-flat sheets of silicon-based membranes for implantable or portable hemodialysis applications. The silicon nanopore membranes (SNM) have biomimetic slit-pore geometry and uniform pores size distribution that allow for exceptional permeability and selectivity. A quantitative diffusion model identified structural limits to diffusive solute transport and motivated a new microfabrication technique to create SNM with enhanced diffusive transport. We performed in vitro testing and extracorporeal testing in pigs on prototype membranes with an effective surface area of 2.52 cm2 and 2.02 cm2, respectively. The diffusive clearance was a two-fold improvement in with the new microfabrication technique and was consistent with our mathematical model. These results establish the feasibility of using SNM for hemodialysis applications with additional scale-up. PMID:27438878

Isolation and metagenomic characterization of bacteria associated with calcium carbonate and struvite precipitation in a pure moving bed biofilm reactor-membrane bioreactor.

PubMed

Gonzalez-Martinez, A; Leyva-Díaz, J C; Rodriguez-Sanchez, A; Muñoz-Palazon, B; Rivadeneyra, A; Poyatos, J M; Rivadeneyra, M A; Martinez-Toledo, M V

2015-01-01

A bench-scale pure moving bed bioreactor-membrane bioreactor (MBBR-MBR) used for the treatment of urban wastewater was analyzed for the identification of bacterial strains with the potential capacity for calcium carbonate and struvite biomineral formation. Isolation of mineral-forming strains on calcium carbonate and struvite media revealed six major colonies with a carbonate or struvite precipitation capacity in the biofouling on the membrane surface and showed that heterotrophic bacteria with the ability to precipitate calcium carbonate and struvite constituted ~7.5% of the total platable bacteria. These belonged to the genera Lysinibacillus, Trichococcus, Comamomas and Bacillus. Pyrosequencing analysis of the microbial communities in the suspended cells and membrane biofouling showed a high degree of similarity in all the samples collected with respect to bacterial assemblage. The study of operational taxonomic units (OTUs) identified through pyrosequencing suggested that ~21% of the total bacterial community identified in the biofouling could potentially form calcium carbonate or struvite crystals in the pure MBBR-MBR system used for the treatment of urban wastewater.

Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes

PubMed Central

Pfister, Klaus; Steinback, Katherine E.; Gardner, Gary; Arntzen, Charles J.

1981-01-01

2-Azido-4-ethylamino-6-isopropylamino-s-triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido[14C]atrazine resulted in the covalent attachment of radioactive inhibitor to thylakoid membranes isolated from pea seedlings and from a triazine-susceptible biotype of the weed Amaranthus hybridus. No covalent binding of azido-atrazine was observed for thylakoid membranes isolated from a naturally occurring triazine-resistant biotype of A. hybridus. Analysis of thylakoid polypeptides from both the susceptible and resistant A. hybridus biotypes by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by fluorography to locate 14C label, demonstrated specific association of the azido[14C]atrazine with polypeptides of the 34- to 32-kilodalton size class in susceptible but not in resistant membranes. Images PMID:16592984

Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes

PubMed Central

Yaakoubi, Hnia; Hamdani, Saber; Bekalé, Laurent; Carpentier, Robert

2014-01-01

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O2 uptake rates, P700 photooxidation and the accumulation of superoxide anions (O2 −) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O2 − generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O2 − generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer. PMID:25420109

A Developmental Study of Photosystem I Peripheral Chlorophyll Proteins 1

PubMed Central

Mullet, John E.; Burke, John J.; Arntzen, Charles J.

1980-01-01

An isolated “native” photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae (≃45 Chl/P700) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association. Chloroplasts were isolated from a Chl b-less barley mutant and from partially-developed cucumber cotyledons (greened under intermittent illumination [ImL] chloroplasts) and were compared to control chloroplasts isolated from wild-type barley and mature cucumber. Both the mutant and ImL chloroplasts exhibited a long wavelength fluorescence maximum at 724 nanometers at 77 K as compared to 735 to 738 nanometers emission maximum in the respective controls. Both the mutant and ImL chloroplasts were deficient in polypeptides of 21,500 to 24,500 daltons which were present in control membranes and in PSI fractions isolated from control membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range paralleled the appearance of PSI Chl species fluorescing at long wavelength (≃735 nm). The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 Chl/P700 (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus ≃ 45 Chl/P700. The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts. Images PMID:16661289

A MEMBRANE FILTER PROCEDURE FOR ASSAYING CYTOTOXIC ACTIVITY IN HETEROTROPHIC BACTERIA ISOLATED FROM DRINKING WATER

EPA Science Inventory

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Wate...

Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2013-01-15

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Apel, William A [Jackson, WY; Thompson, Vicki S [Idaho Falls, ID; Reed, David W [Idaho Falls, ID; Lacey, Jeffrey A [Idaho Falls, ID

2011-12-06

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic sugar transporter genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Apel, William A [Jackson, WY; Thompson, Vicki S [Idaho Falls, ID; Reed, David W [Idaho Falls, ID; Lacey, Jeffrey A [Idaho Falls, ID

2011-06-14

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2013-01-29

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A

2013-11-05

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates.

PubMed

Lavigne, Jean-Philippe; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène; Bouziges, Nicole; Pagès, Jean-Marie; Davin-Regli, Anne

2013-02-01

Imipenem (IPM) is a carbapenem antibiotic frequently used in severe hospital infections. Several reports have mentioned the emergence of resistant isolates exhibiting membrane modifications. A study was conducted between September 2005 and August 2007 to survey infections due to Enterobacter aerogenes in patients hospitalised in a French university hospital. Resistant E. aerogenes clinical isolates obtained from patients treated with IPM and collected during the 3 months following initiation of treatment were phenotypically and molecularly characterised for β-lactamases, efflux pumps activity and outer membrane proteins. Among the 339 patients infected with E. aerogenes during the study period, 41 isolates (12.1%) were resistant to extended-spectrum cephalosporins and 17 patients (5.0%) were treated with IPM. The isolates from these 17 patients presented TEM-24 and basal efflux expression. Following IPM treatment, an IPM-intermediate-susceptible (IPM-I) isolate emerged in 11 patients and an IPM-resistant (IPM-R) isolate in 6 patients. A change in the porin balance (Omp35/Omp36) was observed in IPM-I isolates exhibiting ertapenem resistance. Finally, a porin deficiency (Omp35 and Omp36 absence) was detected in IPM-R isolates associated with efflux pump expression. This study indicates that the alteration in porin expression, including the shift of porin expression and lack of porins, contribute to the E. aerogenes adaptive response to IPM treatment. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

A technique for detecting matrix proteins in the crystalline spicule of the sea urchin embryo.

PubMed

Cho, J W; Partin, J S; Lennarz, W J

1996-02-06

The presence of proteins associated with the CaCO3-containing biocrystals found in a wide variety of marine organisms is well established. In these organisms, including the primitive skeleton (spicule) of the sea urchin embryo, the structural and functional role of these proteins either in the biomineralization process or in control of the structural features of the biocrystals is unclear. Recently, one of the matrix proteins of the sea urchin spicule, SM 30, has been shown to contain a carbohydrate chain (the 1223 epitope) that has been implicated in the process whereby Ca2+ is deposited as CaCo3. Because an understanding of the localization of this protein, as well as other proteins found within the spicule, is central to understanding their function, we undertook to develop methods to localize spicule matrix proteins in intact spicules, using immunogold techniques and scanning electron microscopy. Gold particles indicative of this matrix glycoprotein could not be detected on the surface of spicules that had been isolated from embryo homogenates and treated with alkaline hypochlorite to remove any associated membranous material. However, when isolated spicules were etched for 2 min with dilute acetic acid (10 mM) to expose more internal regions of the crystal, SM 30 and perhaps other proteins bearing the 1223 carbohydrate epitope were detected in the calcite matrix. These results, indicating that these two antigens are widely distributed in the spicule, suggest that this technique should be applicable to any matrix protein for which antibodies are available.

Mechanism and structure of the plant plasma membrane Ca{sup 2+}-ATPase

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

Briskin, D.P.

1993-12-31

Objectives of this project were the following: development of an enriched preparation of the red beet plasma membrane Ca{sup 2+} ATPase in order to develop a procedure for detergent solubilization of the enzyme from the membrane using detergents, resolution by a method which could be upscaled for batch isolation, and then reconstitution into liposomes to allow characterization of Ca{sup 2+} transport by the purified enzyme and; characterization of the reaction mechanism for the coupling of nucleoside triphosphate hydrolysis to Ca{sup 2+} transport as mediated by the plasma membrane Ca{sup 2+} ATPase.

Solid and Liquid Waste Drying Bag

NASA Technical Reports Server (NTRS)

Litwiller, Eric (Inventor); Hogan, John A. (Inventor); Fisher, John W. (Inventor)

2009-01-01

Method and system for processing waste from human activities, including solids, liquids and vapors. A fluid-impermeable bag, lined with a liquid-impermeable but vapor-permeable membrane, defining an inner bag, is provided. A vacuum force is provided to extract vapors so that the waste is moved toward a selected region in the inner bag, extracted vapors, including the waste vapors and vaporized portions of the waste liquids are transported across the membrane, and most or all of the solids remain within the liner. Extracted vapors are filtered, and sanitized components thereof are isolated and optionally stored. The solids remaining within the liner are optionally dried and isolated for ultimate disposal.

Design and simulation of RF MEMS SPST shunt and SPDT shunt-shunt switches for X-band and Ku-band applications

NASA Astrophysics Data System (ADS)

Lenka, Manas K.; Sharma, Amit; Sharma, Jaibir; DasGupta, Amitava

2012-10-01

This paper describes the design and simulation of RF MEMS SPST shunt and SPDT shunt-shunt switches with modified coplanar waveguide (CPW) configuration for X-band and Ku-band applications exhibiting high isolation and low insertion loss. By modifying the basic CPW structure for a six-strip membrane having length 720 μm, the resonant frequency can be reduced from 33.5 GHz to 13.5 GHz with isolation as high as -30 dB(-63 dB at resonant frequency) in Ku-band. Similar results are also found in case SPST and SPDT switches with other membrane types.

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

PubMed

Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

2018-01-01

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria. Copyright © 2017. Published by Elsevier Inc.

Evaluation of periosteum eversion and coronally advanced flap techniques in the treatment of isolated Miller's Class I/II gingival recession: A comparative clinical study

PubMed Central

Debnath, Koel; Chatterjee, Anirban

2018-01-01

Aim: The present investigation aimed to evaluate root coverage (RC) with periosteum eversion technique (PET) using periosteum as a graft and coronally advanced flap (CAF) with platelet-rich fibrin (PRF) membrane as a graft in the treatment of isolated Miller's class I and II gingival recession defects. Materials and Methods: Thirty sites in 15 participants with Miller's Class I or II gingival recession were randomly treated either with PET using periosteum as graft and CAF + PRF as graft. In a split mouth design, the parameters such as recession depth, recession width at cementoenamel junction, probing depth, periodontal attachment level (PAL), and keratinized gingival width were assessed at baseline, 3 months, and 6 months postoperative follow-up with William's graduated probe and Vernier caliper. Results: Both the treatment modalities yielded statistically nonsignificant desirable treatment outcomes at both postoperative levels in terms of all the parameters The mean RC with probe method and Vernier method in CAF + PRF was 75.01% and 86.86%, respectively, and PET showed a mean RC of 61.112% and 83.971%, respectively, at 6-month interval period which showed a nonstatistically significant difference. Conclusion: Both the treatment modalities, i.e., CAF + PRF and PET are essentially and equally effective in the treatment of Miller's Class I or II gingival recession defects. PMID:29769769

Composite zeolite membranes

DOEpatents

Nenoff, Tina M.; Thoma, Steven G.; Ashley, Carol S.; Reed, Scott T.

2002-01-01

A new class of composite zeolite membranes and synthesis techniques therefor has been invented. These membranes are essentially defect-free, and exhibit large levels of transmembrane flux and of chemical and isotopic selectivity.

Antigenic specificity and morphologic characteristics of Chlamydia trachomatis, strain SFPD, isolated from hamsters with proliferative ileitis.

PubMed

Fox, J G; Stills, H F; Paster, B J; Dewhirst, F E; Yan, L; Palley, L; Prostak, K

1993-10-01

Profound diarrhea associated with proliferating intestinal cells containing intraepithelial campylobacter-like organisms (ICLO) occurs in a variety of mammalian hosts, particularly swine and hamsters. Recently, intracellular bacteria were isolated from proliferative intestinal tissue of hamsters and propagated in intestine cell line 407. Oral inoculation of hamsters with cell culture lysates containing these organisms reproduced the disease in susceptible hamsters. In the present study, an intracellular bacterium from the INT 407 cell line was shown by a variety of techniques to be a member of the genus Chlamydia and has been designated Chlamydia sp. strain SFPD. McCoy cells infected with Chlamydia sp. strain SFPD demonstrated bright fluorescent-stained intracytoplasmic inclusions when examined with fluorescein-labeled species-specific C. trachomatis monoclonal antibodies. The organism also reacted to fluorescein-labeled polyclonal but not monoclonal ICLO "omega" antisera. Ultrastructural examination of the Chlamydia sp. strain SFPD from McCoy cells revealed electrondense elementary bodies and a less electron-dense reticulate-like body that was circular; both features are consistent in morphology to developmental forms of Chlamydia and do not conform to ICLO morphology. Molecular studies, 16S ribosomal sequence analysis, and sequencing of the outer membrane protein confirmed that the isolate is a C. trachomatis closely related to the mouse pneumonitis strain of C. trachomatis.

Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol

NASA Astrophysics Data System (ADS)

Livshts, Mikhail A.; Khomyakova, Elena; Evtushenko, Evgeniy G.; Lazarev, Vassili N.; Kulemin, Nikolay A.; Semina, Svetlana E.; Generozov, Edward V.; Govorun, Vadim M.

2015-11-01

Exosomes, small (40-100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors. For both types of rotors - “swinging bucket” and “fixed-angle” - we express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

PubMed

Soejima, Takashi; Xiao, Jin-Zhong; Abe, Fumiaki

2016-06-23

Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

Efficient entrapment of large and small compounds during vesiculation of intestinal microvilli.

PubMed Central

van Dommelen, F S; Hamer, C M; De Jonge, H R

1986-01-01

An efficient method is described permitting the encapsulation of membrane-impermeable compounds at the interior of intestinal microvilli during vesicle formation. Rat intestinal epithelial cells were isolated by high-frequency vibration and exposed transiently to iso-osmotic medium containing 5 mM-EDTA. Vesiculation of microvilli was effected by freeze-thawing instead of mechanical fragmentation or hypo-osmotic lysis. Solutes to be entrapped were mixed with the extracellular medium before freezing in liquid N2. Microvillous vesicles were isolated from thawed cell suspensions by Ca2+- or Mg2+-aggregation of contaminants and differential centrifugation. The yield, purity, orientation and transport properties of the vesicles were similar, or superior, to preparations described in the literature. A high loading efficiency was demonstrated for small impermeants (cyclic GMP, ATP, Arsenazo III) as well as proteins (albumin); in contrast, loading of isolated vesicles by hypo-osmotic shock was only partially effective (cyclic GMP, ATP) or ineffective (albumin). Entrapment of an ATP-regenerating system could partially block a Mg2+-dependent conversion of intravesicular ATP into ADP. No evidence was obtained for the contribution of a proton pump to the intrinsic Mg2+-ATPase of the vesicle. Potential applications of the vesicle-loading technique in studies of brush-border transport regulation by intramicrovillar factors are discussed. Images Fig. 1. Fig. 2. PMID:3024625

Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

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

Montaigne, David; Marechal, Xavier; Baccouch, Riadh

2010-05-01

The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solutionmore » containing 1 muM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt{sub max} of 105 +- 8 mN/s in control hearts vs. 49 +- 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 +- 0.2 in control hearts vs. 2.2 +- 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 +- 1 muM cytochrome c/min/mg in control hearts vs. 14 +- 3 muM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.« less

Folded membrane dialyzer with mechanically sealed edges

DOEpatents

Markley, Finley W.

1976-01-01

A semipermeable membrane is folded in accordion fashion to form a stack of pleats and the edges are sealed so as to isolate the opposite surfaces of the membrane. The stack is contained within a case that provides ports for flow of blood in contact with one surface of the membrane through channels formed by the pleats and also provides ports for flow of a dialysate through channels formed by the pleats in contact with the other surface of the membrane. The serpentine side edges of the membrane are sealed by a solidified plastic material, whereas effective mechanical means are provided to seal the end edges of the folded membrane. The mechanical means include a clamping strip which biases case sealing flanges into a sealed relationship with end portions of the membrane near the end edges, which portions extend from the stack and between the sealing flanges.

A novel bioactive membrane by cell electrospinning.

PubMed

Chen, Haiping; Liu, Yuanyuan; Hu, Qingxi

2015-11-01

Electrospinning permits fabrication of biodegradable matrices that can resemble the both scale and mechanical behavior of the native extracellular matrix. However, achieving high-cellular density and infiltration of cells within matrices with traditional technique remain challenging and time consuming. The cell electrospinning technique presented in this paper can mitigate the problems associated with these limitations. Cells encapsulated by the material in the cell electrospinning technique survived well and distributed homogenously within the nanofibrous membrane, and their vitality was improved to 133% after being cultured for 28 days. The electrospun nanofibrous membrane has a certain degradation property and favorable cell-membrane interaction that supports the active biocompatibility of the membrane. Its properties are helpful for supporting cell attachment and growth, maintaining phenotypic shape, and secreting an ample amount of extracellular matrix (ECM). This novel membrane may be a potential application within the field of tissue engineering. The ability of cell electrospinning to microintegrate cells into a biodegradable fibrous matrix embodies a novel tissue engineering approach that could be applied to fabricate a high cell density elastic tissue mimetic. Copyright © 2015 Elsevier Inc. All rights reserved.

Recent Advances in Nanodisc Technology for Membrane Proteins Studies (2012–2017)

PubMed Central

Rouck, John; Krapf, John; Roy, Jahnabi; Huff, Hannah; Das, Aditi

2017-01-01

Historically, progress in membrane protein research has been hindered due to solubility issues. The introduction of biomembrane mimetics has since stimulated the field’s momentum. One mimetic, the nanodisc, has proved to be an exceptional system for solubilizing membrane proteins. Herein, we critically evaluate the advantages and imperfections from employing nanodiscs in biophysical and biochemical studies. Specifically, we examine the techniques that have been modified to study membrane proteins in nanodiscs. Techniques discussed include fluorescence microscopy, solution state/solid state NMR, electron microscopy, SAXS, and several mass spectroscopy methods. Newer techniques such as SPR, charge sensitive optical detection, and scintillation proximity assays are also reviewed. Lastly, we cover nanodiscs advancing nanotechnology through nanoplasmonic biosensing, lipoprotein-nanoplatelets, and sortase-mediated labeling of nanodiscs. PMID:28581067

Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle.

PubMed

Muñoz, P; Rosemblatt, M; Testar, X; Palacín, M; Zorzano, A

1995-04-01

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy.

PubMed

Joseph, Benesh; Sikora, Arthur; Bordignon, Enrica; Jeschke, Gunnar; Cafiso, David S; Prisner, Thomas F

2015-05-18

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin-labeled in whole cells and outer membranes and interspin distances were measured to a spin-labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

Senning, Eric N.; Aman, Teresa K.

2016-01-01

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

Herpes simplex virus 1 induces de novo phospholipid synthesis

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

Sutter, Esther; Oliveira, Anna Paula de; Tobler, Kurt

2012-08-01

Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [{sup 3}H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [{sup 3}H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and thatmore » de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.« less

Membrane shape modulates transmembrane protein distribution.

PubMed

Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

2014-01-27

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

PubMed Central

Le Gall, Sylvie; Neuhof, Andrea; Rapoport, Tom

2004-01-01

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of ∼5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER. PMID:14617815

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.

Novel three-dimensional autologous tissue-engineered vaginal tissues using the self-assembly technique.

PubMed

Orabi, Hazem; Saba, Ingrid; Rousseau, Alexandre; Bolduc, Stéphane

2017-02-01

Many diseases necessitate the substitution of vaginal tissues. Current replacement therapies are associated with many complications. In this study, we aimed to create bioengineered neovaginas with the self-assembly technique using autologous vaginal epithelial (VE) and vaginal stromal (VS) cells without the use of exogenous materials and to document the survival and incorporation of these grafts into the tissues of nude female mice. Epithelial and stromal cells were isolated from vaginal biopsies. Stromal cells were driven to form collagen sheets, 3 of which were superimposed to form vaginal stromas. VE cells were seeded on top of these stromas and allowed to mature in an air-liquid interface. The vaginal equivalents were implanted subcutaneously in female nude mice, which were sacrificed after 1 and 2 weeks after surgery. The in vitro and animal-retrieved equivalents were assessed using histologic, functional, and mechanical evaluations. Vaginal equivalents could be handled easily. VE cells formed a well-differentiated epithelial layer with a continuous basement membrane. The equivalent matrix was composed of collagen I and III and elastin. The epithelium, basement membrane, and stroma were comparable to those of native vaginal tissues. The implanted equivalents formed mature vaginal epithelium and matrix that were integrated into the mice tissues. Using the self-assembly technique, in vitro vaginal tissues were created with many functional and biological similarities to native vagina without any foreign material. They formed functional vaginal tissues after in vivo animal implantation. It is appropriate for vaginal substitution and disease modeling for infectious studies, vaginal applicants, and drug testing. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.

PubMed Central

Dzandu, J K; Deh, M E; Barratt, D L; Wise, G E

1984-01-01

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes. Images PMID:6200882

Langmuir-Blodgett and X-ray diffraction studies of isolated photosystem II reaction centers in monolayers and multilayers: physical dimensions of the complex.

PubMed

Uphaus, R A; Fang, J Y; Picorel, R; Chumanov, G; Wang, J Y; Cotton, T M; Seibert, M

1997-04-01

The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2 and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. Pl. Physiol. 22, 161-166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.

Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats.

PubMed

Roman-Campos, D; Campos, A C; Gioda, C R; Campos, P P; Medeiros, M A A; Cruz, J S

2009-06-05

Thiamine is an important cofactor present in many biochemical reactions, and its deprivation can lead to heart dysfunction. Little is known about the influence of thiamine deprivation on the electrophysiological behavior of the isolated heart cells and information about thiamine deficiency in heart morphology is controversial. Thus, we decided to investigate the major repolarizing conductances and their influence in the action potential (AP) waveform as well as the changes in the heart structure in a set of thiamine deficiency in rats. Using the patch-clamp technique, we investigated inward (I(K1)) and outward K(+) currents (I(to)), T-type and L-type Ca(2+) currents and APs. To evaluate heart morphology we used hematoxylin and eosin in transversal heart sections. Thiamine deficiency caused a marked decrease in left ventricle thickness, cardiomyocyte number, cell length and width, and membrane capacitance. When evaluating I(to) we did not find difference in current amplitude; however an acceleration of I(to) inactivation was observed. I(K1) showed a reduction in the amplitude and slope conductance, which implicated a less negative resting membrane potential in cardiac myocytes isolated from thiamine-deficient rats. We did not find any difference in L-type Ca(2+) current density. T-type Ca(2+) current was not observed. In addition, we did not observe significant changes in AP repolarization. Based on our study we can conclude that thiamine deficiency causes heart hypotrophy and not heart hypertrophy. Moreover, we provided evidence that there is no major electrical remodeling during thiamine deficiency, a feature of heart failure models.

Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity.

PubMed

Kuo, Mario Meng-Chiang; Baker, Kent A; Wong, Lee; Choe, Senyon

2007-02-13

The crystal structure of the RCK-containing MthK provides a molecular framework for understanding the ligand gating mechanisms of K+ channels. Here we examined the macroscopic currents of MthK in enlarged Escherichia coli membrane by patch clamp and rapid perfusion techniques and showed that the channel undergoes desensitization in seconds after activation by Ca2+ or Cd2+. Additionally, MthK is inactivated by slightly acidic pH only from the cytoplasmic side. Examinations of isolated RCK domain by size-exclusion chromatography, static light scattering, analytical sedimentation, and stopped-flow spectroscopy show that Ca2+ rapidly converts isolated RCK monomers to multimers at alkaline pH. In contrast, the RCK domain at acidic pH remains firmly dimeric regardless of Ca2+ but restores predominantly to multimer or monomer at basic pH with or without Ca2+, respectively. These functional and biochemical analyses correlate the four functional states of the MthK channel with distinct oligomeric states of its RCK domains and indicate that the RCK domains undergo oligomeric conversions in modulating MthK activities.

Markers of feline leukaemia virus infection or exposure in cats from a region of low seroprevalence.

PubMed

Beatty, Julia A; Tasker, Séverine; Jarrett, Oswald; Lam, Amy; Gibson, Stephanie; Noe-Nordberg, Alice; Phillips, Angela; Fawcett, Anne; Barrs, Vanessa R

2011-12-01

Molecular techniques have demonstrated that cats may harbour feline leukaemia virus (FeLV) provirus in the absence of antigenaemia. Using quantitative real-time polymerase chain reaction (qPCR), p27 enzyme-linked immunosorbent assay (ELISA), anti-feline oncornavirus-associated cell-membrane-antigen (FOCMA) antibody testing and virus isolation (VI) we investigated three groups of cats. Among cats with cytopenias or lymphoma, 2/75 were transiently positive for provirus and anti-FOCMA antibodies were the only evidence of exposure in another. In 169 young, healthy cats, all tests were negative. In contrast, 3/4 cats from a closed household where FeLV was confirmed by isolation, had evidence of infection. Our results support a role for factors other than FeLV in the pathogenesis of cytopenias and lymphoma. There was no evidence of exposure in young cats. In regions of low prevalence, where the positive predictive value of antigen testing is low, qPCR may assist with diagnosis. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

PubMed

Onken, Horst; Patel, Malay; Javoroncov, Margarita; Izeirovski, Sejmir; Moffett, Stacia B; Moffett, David F

2009-03-01

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

Examination of mitral regurgitation with a goat heart model for the development of intelligent artificial papillary muscle.

PubMed

Shiraishi, Y; Yambe, T; Yoshizawa, M; Hashimoto, H; Yamada, A; Miura, H; Hashem, M; Kitano, T; Shiga, T; Homma, D

2012-01-01

Annuloplasty for functional mitral or tricuspid regurgitation has been made for surgical restoration of valvular diseases. However, these major techniques may sometimes be ineffective because of chamber dilation and valve tethering. We have been developing a sophisticated intelligent artificial papillary muscle (PM) by using an anisotropic shape memory alloy fiber for an alternative surgical reconstruction of the continuity of the mitral structural apparatus and the left ventricular myocardium. This study exhibited the mitral regurgitation with regard to the reduction in the PM tension quantitatively with an originally developed ventricular simulator using isolated goat hearts for the sophisticated artificial PM. Aortic and mitral valves with left ventricular free wall portions of isolated goat hearts (n=9) were secured on the elastic plastic membrane and statically pressurized, which led to valvular leaflet-papillary muscle positional change and central mitral regurgitation. PMs were connected to the load cell, and the relationship between the tension of regurgitation and PM tension were measured. Then we connected the left ventricular specimen model to our hydraulic ventricular simulator and achieved hemodynamic simulation with the controlled tension of PMs.

Evidence for a Na+/H+ Antiporter in Membrane Vesicles Isolated from Roots of the Halophyte Atriplex nummularia1

PubMed Central

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

1988-01-01

The ATP-dependent establishment of a positive membrane potential (measured as S14CN−-accumulation) in membrane vesicles isolated from the roots of Atriplex nummularia Lindl. was not inhibited by NaMes and KMes at concentrations up to 140 millimolar. On the other hand, the formation of ΔpH (measured as 14C-methylamine accumulation or quenching of quinacrine fluorescence), was depressed by NaMes concentrations as low as 30 millimolar. Supply of NaMes after the ΔpH had been established brought about partial dissipation within 30 seconds. Extent of dissipation of ΔpH increased with NaMes concentration over the range tested (up to 180 millimolar). The H+/Na+ exchange indicated by these results was not due to the creation of a Na+ diffusion potential. Formation of ΔpH in these vesicles was stable to NO3− up to 100 millimolar; further, the dissipating effect of Na+ supply was apparent on a ΔpH formed in the presence of 30 millimolar NO3−. Additional evidence that the origin of the membrane vesicles observed in this investigation was not the tonoplast and was probably the plasmalemma included the vanadate sensitivity of the establishment of the membrane potential. PMID:16666082

Mitigation of Membrane Biofouling in MBR Using a Cellulolytic Bacterium, Undibacterium sp. DM-1, Isolated from Activated Sludge.

PubMed

Nahm, Chang Hyun; Lee, Seonki; Lee, Sang Hyun; Lee, Kibaek; Lee, Jaewoo; Kwon, Hyeokpil; Choo, Kwang-Ho; Lee, Jung-Kee; Jang, Jae Young; Lee, Chung-Hak; Park, Pyung-Kyu

2017-03-28

Biofilm formation on the membrane surface results in the loss of permeability in membrane bioreactors (MBRs) for wastewater treatment. Studies have revealed that cellulose is not only produced by a number of bacterial species but also plays a key role during formation of their biofilm. Hence, in this study, cellulase was introduced to a MBR as a cellulose-induced biofilm control strategy. For practical application of cellulase to MBR, a cellulolytic ( i.e ., cellulase-producing) bacterium, Undibacterium sp. DM-1, was isolated from a lab-scale MBR for wastewater treatment. Prior to its application to MBR, it was confirmed that the cell-free supernatant of DM-1 was capable of inhibiting biofilm formation and of detaching the mature biofilm of activated sludge and cellulose-producing bacteria. This suggested that cellulase could be an effective anti-biofouling agent for MBRs used in wastewater treatment. Undibacterium sp. DM-1-entrapping beads ( i.e ., cellulolytic-beads) were applied to a continuous MBR to mitigate membrane biofouling 2.2-fold, compared with an MBR with vacant-beads as a control. Subsequent analysis of the cellulose content in the biofilm formed on the membrane surface revealed that this mitigation was associated with an approximately 30% reduction in cellulose by cellulolytic-beads in MBR.

Interaction of sodium dodecyl sulfate with watermelon chromoplasts and examination of the organization of lycopene within the chromoplasts.

PubMed

Fish, Wayne W

2006-10-18

The properties of plant-derived precipitates of watermelon lycopene were examined in aqueous sodium dodecyl sulfate (SDS) as part of an ongoing effort to develop simpler, more economical ways to quantify carotenoids in melon fruit. Levels of SDS >0.2% were found to increase the water solubility of lycopene in the state in which it was isolated from watermelon. Electron microscopy and chemical analyses suggested that the watermelon lycopene as isolated is packaged inside a membrane to form a chromoplast. Spectral peaks in the visible region of the watermelon chromoplasts in SDS exhibited a bathochromic shift from those in organic solvent. Watermelon chromoplasts in SDS exhibited pronounced circular dichroic activity in the visible region. Binding measurements indicated that about 120 molecules of SDS were bound per molecule of lycopene inside the chromoplast; likely, the detergent molecules are bound to the chromoplast membrane. Around 80% of the chromoplast-SDS complexes were retained on a 0.45 mum membrane filter. Together, these observations are consistent with lycopene in a J-type chiral arrangement inside a membrane to form a chromoplast. The binding of SDS molecules to the chromoplast membrane form a complex that is extensively more water-soluble than the chromoplast alone.

Fabrication of composite membranes using copper metal organic framework for energy application

NASA Astrophysics Data System (ADS)

Gahlot, Swati; Rajput, Abhishek; Kulshrestha, Vaibhav

2018-04-01

Present manuscript deals with the synthesis of nanocomposite polymer electrolyte membrane (PEM) based on copper based metal organic framework (Cu-MOF) and sulfonated poly ether sulfone (SPES) for fuel cell application. Prepared material and composite membrane has been analyzed through various techniques. Structural and thermal characterization of prepared material has been carried out through XRD, FTIR and TGA technique. Measurement shows the successful synthesis of MOF and also confirms the thermal stability. Prepared membranes shows good physicochemical properties and good ionic conductivity which can be utilized as PEM for fuel cell application.

Analysis of the interaction between membrane proteins and soluble binding partners by surface plasmon resonance.

PubMed

Wu, Zht Cheng; de Keyzer, Jeanine; Kusters, Ilja; Driessen, Arnold J M

2013-01-01

The interaction between membrane proteins and their (protein) ligands is conventionally investigated by nonequilibrium methods such as co-sedimentation or pull-down assays. Surface Plasmon Resonance can be used to monitor such binding events in real-time using isolated membranes immobilized to a surface providing insights in the kinetics of binding under equilibrium conditions. This application provides a fast, automated way to detect interacting species and to determine the kinetics and affinity (Kd) of the interaction.