scholarly journals Separation of Membrane Vesicles and Cytosol from Yeast, Cultured Cells, and Bacteria in a Small Volume Self-Generated Gradient in a Fixed-Angle Rotor

2002 ◽  
Vol 2 ◽  
pp. 1603-1606
Author(s):  
John Graham
Keyword(s):  
Opposite Direction ◽  
Small Volume ◽  
Cultured Cells ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Sample Zone ◽  
Fixed Angle ◽  
Low Viscosity

There are many situations when it is necessary to separate rapidly and efficiently a cytosolic and a membrane vesicle fraction from yeast, cultured cells, or from bacteria. This Protocol Article describes the flotation of the vesicles through a self-generated gradient from a dense sample zone using the low-viscosity medium iodixanol. As the sample is exposed to the gmaxthe tendency of the proteins to sediment overcomes any diffusion in the opposite direction and are therefore completely separated from the vesicles.

scholarly journals Separation of Membrane Vesicles and Cytosol from Cultured Cells and Bacteria in a Preformed Discontinuous Gradient

2002 ◽  
Vol 2 ◽  
pp. 1555-1559 ◽  
Author(s):  
John Graham
Keyword(s):  
Opposite Direction ◽  
Cultured Cells ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Complete Separation ◽  
Low Density ◽  
Sample Zone ◽  
Low Viscosity ◽  
Density Barrier

There are many situations when it is necessary to separate rapidly and efficiently a cytosolic and a membrane vesicle fraction from either cultured cells or from bacteria. Flotation of the vesicles through a low-density barrier from a dense sample zone using the low viscosity medium iodixanol allows complete separation of these compartments. As the sample is exposed to the gmax the tendency of the proteins to sediment overcomes any diffusion in the opposite direction.

scholarly journals Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce

2020 ◽  
Vol 71 (20) ◽  
pp. 6379-6395 ◽  
Author(s):  
Enni Väisänen ◽  
Junko Takahashi ◽  
Ogonna Obudulu ◽  
Joakim Bygdell ◽  
Pirkko Karhunen ◽  
...  
Keyword(s):  
Norway Spruce ◽  
Cultured Cells ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Monolignol Biosynthesis ◽  
Nicotiana Tabacum L ◽  
Km Value ◽  
Mfs Transporters

Abstract Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

scholarly journals Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro

2021 ◽  
Vol 22 (6) ◽  
pp. 3163
Author(s):  
Hirofumi Ohashi ◽  
Feng Wang ◽  
Frank Stappenbeck ◽  
Kana Tsuchimoto ◽  
Chisa Kobayashi ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Cultured Cells ◽  
Peak Plasma ◽  
Plasma Concentrations ◽  
Membrane Vesicles ◽  
Drug Candidates ◽  
Increased Risk ◽  
Derivatives Of

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

Immunogold-Silver Staining (IGSS) of (NCP and CP) BVDV Infected Subcellular Fraction Bands

1997 ◽  
Vol 3 (S2) ◽  
pp. 155-156
Author(s):  
C.E. Hearne ◽  
D.L. Johnson ◽  
H. Van Campen
Keyword(s):  
Subcellular Fraction ◽  
Membrane Vesicles ◽  
Bovine Viral Diarrhea ◽  
Fixed Angle ◽  
Cell Monolayers ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Transmission Electron ◽  
Viral Diarrhea Virus ◽  
Bovine Turbinate

Pre-embedding immunogold-silver (IGSS) techniques are useful to localize antigens in cell monolayers and agarose embedded cell suspensions for transmission electron microscopy. Procedural centrifugations, however, present a challenge when attempting to localize antigens in subcellular fractions. Using a Beckman Airfuge Ultracentrifuge to concentrate the subcellar fraction bands and resuspending the organelles in agarose simplifies IGSS processing and resin embedding procedures.Control bovine turbinate (BT), and BT cells infected with cytopathic (cp NADL) and non-cytopathic (ncp NY-1) strains of bovine viral diarrhea virus (BVDV) were fractioned according to Bienz et al (1992). Bands containing membrane vesicles (Fig 2) were collected and each fraction band was pelleted at 169,000g for 20 min using an A-95 fixed angle rotor in a Beckman Airfuge Ultracentrifuge. Each fraction pellet was resuspended in 50μl of 30% agarose, solidified, and trimmed to < lmm. IGSS procedures were carried out according to Nanoprobes, Inc., Stoney Brook, NY, and Hearne & Van Campen (1996).

scholarly journals Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1661
Author(s):  
Mei-Hsiu Chen ◽  
Tse-Ying Liu ◽  
Yu-Chiao Chen ◽  
Ming-Hong Chen
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Glioma Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
E Coli ◽  
Tumor Bearing ◽  
Nano Gold ◽  
Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

scholarly journals Membrane Vesicle Release in Bacteria, Eukaryotes, and Archaea: a Conserved yet Underappreciated Aspect of Microbial Life

2012 ◽  
Vol 80 (6) ◽  
pp. 1948-1957 ◽  
Author(s):  
Brooke L. Deatherage ◽  
Brad T. Cookson
Keyword(s):  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Immune System ◽  
Vesicle Release ◽  
Communication Signals ◽  
Microbial Life ◽  
Functional Features

ABSTRACTInteraction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not onlyin vitrobut alsoin vivoduring infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.

The iodide channel of the thyroid: a plasma membrane vesicle study

1992 ◽  
Vol 263 (3) ◽  
pp. C590-C597 ◽  
Author(s):  
P. Golstein ◽  
M. Abramow ◽  
J. E. Dumont ◽  
R. Beauwens
Keyword(s):  
Plasma Membrane ◽  
Chloride Transport ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Anion Channel ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Vesicle ◽  
Plasma Membrane Vesicles ◽  
Bovine Thyroid ◽  
Set Up

The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.

scholarly journals Outer Membrane Machinery and Alginate Synthesis Regulators Control Membrane Vesicle Production in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (24) ◽  
pp. 7509-7519 ◽  
Author(s):  
Yosuke Tashiro ◽  
Ryosuke Sakai ◽  
Masanori Toyofuku ◽  
Isao Sawada ◽  
Toshiaki Nakajima-Kambe ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Serine Proteases ◽  
Membrane Vesicle ◽  
Bacterial Pathogen ◽  
Membrane Vesicles ◽  
Production Mechanism ◽  
Gene Encoding ◽  
Surrounding Environment ◽  
Regulatory Machinery

ABSTRACT The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

scholarly journals Phospholipase D2 Localizes to the Plasma Membrane and Regulates Angiotensin II Receptor Endocytosis

2004 ◽  
Vol 15 (3) ◽  
pp. 1024-1030 ◽  
Author(s):  
Guangwei Du ◽  
Ping Huang ◽  
Bruce T. Liang ◽  
Michael A. Frohman
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Dominant Negative ◽  
Secretory Cells ◽  
Resting Cells ◽  
Angiotensin Ii Receptor ◽  
Multiple Cell

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.

Reconstitution of Single Potassium Channels from Bovine Gallbladder Epithelium

1998 ◽  
Vol 26 (4) ◽  
pp. 188-199
Author(s):  
E Kyriacou
Keyword(s):  
Ion Channels ◽  
Gall Bladder ◽  
Lipid Bilayers ◽  
Membrane Vesicle ◽  
Basolateral Membrane ◽  
Molecular Transport ◽  
Membrane Vesicles ◽  
Potassium Ion ◽  
Potassium Ion Channels ◽  
Adult Cattle

The study of molecular transport across gall-bladder epithelium may contribute to our understanding of the pathophysiology of gall-bladder disease. The aim of this study was to reconstitute and characterize single potassium ion channels in bovine gall-bladder epithelial mucosa – both apical and basolateral aspects. Standard subcellular fractionation techniques were used to form either apical or basolateral closed-membrane vesicles from the mucosal epithelium of fresh gall bladders from healthy young adult cattle. Vesicular ion channels were incorporated into voltage-clamped planar lipid bilayers under known ionic conditions and their conductances, reversal potentials, and voltages were characterized. Low-conductance voltage-insensitive apical membrane vesicle channels of at least four conductance levels were found (mean ± SD): 12 ± 4 pS, n = 10; 40 ± 12 pS, n = 4; 273 ± 31 pS, n = 3; and 151 ± 24 pS, n = 5. Conductances of potassium ion channels in basolateral membrane vesicles were in the range 9–450 pS, and these channels included high-conductance calcium-activated potassium-ion channels ‘K(Ca)’ which were voltage- and calcium-dependent.

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