Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “outer membrane vesicles”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed1989

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Animals↗
PubMed1989

Cholesterol movement between bovine rod outer segment disk membranes and phospholipid vesicles.

The ability of cholesterol to move between bovine rod outer segment disk membranes and phospholipid membranes was examined. Disk membranes were incubated with small unilamellar phospholipid vesicles containing varying amounts of cholesterol. Aliquots were removed at specific times, and then the disks and the vesicles were separated by centrifugation and assayed for phospholipid and cholesterol content. When incubated with vesicles containing no cholesterol, the cholesterol to phospholipid ratio in the disk membrane was reduced due to migration of cholesterol from the disks into the vesicles. The cholesterol content of these cholesterol depleted disks could be readily returned to the normal disk cholesterol content by incubation of the cholesterol-depleted disks with small unilamellar vesicles containing high cholesterol. An apparent partition coefficient K was calculated as the quotient of the cholesterol/phospholipid mole ratio in the donor membranes and the cholesterol/phospholipid mole ratio in the acceptor membranes. The value of K was approximately 1 at cholesterol levels below normal disk cholesterol content, for disk membranes and phosphatidylcholine small unilamellar vesicles. Inclusion of phosphatidylethanolamine in the small unilamellar vesicle acceptor raised K, indicating that phosphatidylethanolamine creates an unfavourable environment for cholesterol. The cholesterol to phospholipid ratio of native disks could be increased by incubation with phosphatidylcholine small unilamellar vesicles (donor) which contained higher amounts of cholesterol than the disk membrane acceptor. In these experiments the distribution of cholesterol between disks and small unilamellar vesicles always favored the vesicles. The apparent partition coefficient was 1.7 at several cholesterol levels above the native disk cholesterol content. Liposomes made from lipid extracted from the disk membrane behaved in the same manner as intact disks with respect to cholesterol distribution at equilibrium. The phospholipid content of the disk membrane may be an important factor in determining the cholesterol content of the disk membrane.

Animals↗
PubMed1984

Renal transport of neutral amino acids. Tubular localization of Na+-dependent phenylalanine- and glucose-transport systems.

The transport properties for phenylalanine and glucose in luminal-membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit kidney were studied by a spectrophotometric method. Uptake of phenylalanine as well as of glucose by the two types of membrane vesicles was found to be Na+-dependent, electrogenic and stereospecific. Na+-dependent transport of L-phenylalanine by outer-cortical membrane vesicles could be accounted for by one transport system (KA congruent to 1.5 mM). By contrast, in the outer-medullary preparation, L-phenylalanine transport occurred via two transport systems, namely a high-affinity system with K1A congruent to 0.33 mM and a low-affinity system with K2A congruent to 7 mM respectively. Na+-dependent uptake of D-glucose by pars convoluta and pars recta membrane vesicles could be described by single, but different, transport systems, namely a low-affinity system with KA congruent to 3.5 mM and a high-affinity system with KA congruent to 0.30 mM respectively. Attempts to calculate the stoichiometry of the different Na+/D-glucose transport systems by using Hill-type plots revealed that the ratio of the Na+/hexose co-transport probably is 1:1 in the case of pars convoluta and 2:1 in membrane vesicles from pars recta. The Na+/L-phenylalanine stoichiometry of the pars convoluta transporter probably is 1:1. Both the high-affinity and the low-affinity Na+-dependent L-phenylalanine transport system of pars recta membrane vesicles seem to operate with a 1:1 stoichiometry. The physiological importance of the arrangement of low-affinity and high-affinity transport systems along the kidney proximal tubule is discussed.

Animals↗
PubMed1999

Immunohistochemical demonstration of a neuronal calmodulin-binding protein, NAP-22, in the rat spinal cord.

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a newly isolated calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. To reveal the NAP-22 distribution in vivo, we investigated the spinal cord of the 4-5-week old rats using light and electron microscopy. NAP-22 immunoreactivity was observed in the gray matter with dorsoventral gradient of reactivity. Distinct reactivity was demonstrated in the nerve terminals and dendritic spines. Some reactions were also observed in the thin nerve fibers. NAP-22 immunoreactivity was associated mainly with pre- and postsynaptic membranes, synaptic vesicles and outer mitochondrial membranes. In the nerve terminals, NAP-22 was colocalized with synaptic vesicle proteins such as synapsin I or synaptobrevin 2. About 80% of the nerve terminals having immunoreactivity for synapsin I or synaptobrevin 2 showed NAP-22 immunoreactivity. From these results, NAP-22 is confirmed to be distributed in the synaptic region of the spinal cord and is involved in the synaptic function relating to PKC.

Animals↗
PubMed1990

Profiles of serological reactivity against cytosoluble antigens of Brucella ovis in experimentally infected rams.

Sera from rams infected with and excreting Brucella ovis in the semen (shedders), as well as from animals which had recovered from previous experimental challenge with B. ovis, were analyzed for their serological reactivities against cytosolic antigens of the bacterium. Membrane vesicles, including outer and inner membrane components, were precluded from the analyses by subjecting French-pressed bacteria to ultracentrifugation. The resulting cytosolic supernatant was fractionated into four major antigenic fractions, fractions A, B, C, and D, by high-pressure liquid chromatography. Temporal enzyme-linked immunosorbent assays with the A antigen revealed that all shedder rams displayed a rise-and-surge response, while rams which recovered from experimental challenge showed a rise-and-fall profile. The B antigen was less discriminatory in detecting a difference between the two ram groups, while C and D antigens were serologically unreactive in the enzyme-linked immunosorbent assay. In contrast to the reactivity patterns shown by native high-pressure liquid chromatography-fractionated cytosolic supernatant antigens, immunoblotting of C and D polypeptides generated by boiling in the presence of sodium dodecyl sulfate and mercaptoethanol was particularly useful in distinguishing between sera collected at the mid-surge phase of infected rams from sera obtained at the mid-fall stage of recovered animals. It is likely that native or denatured antigens of different cytosolic fractions may provide useful serological reagents for differentiating between infected rams and those which have recovered from exposure to B. ovis.

Animals↗
PubMed1981

K88-mediated binding of Escherichia coli outer membrane fragments to porcine intestinal epithelial cell brush borders.

We have examined the interactions between various radiolabeled membrane fractions obtained from an enterotoxigenic Escherichia coli strain and brush borders isolated from porcine intestinal epithelial cells. Outer membrane fragments containing the K88 attachment factor bound tightly to brush borders, whereas cytoplasmic membrane vesicles did not. Three different types of outer membrane preparations were tested: (i) cellular outer membranes isolated from lysozyme spheroplasts, (ii) medium vesicles or outer membrane fragments released into the medium during growth, and (iii) periplasmic vesicles, or outer membrane fragments which were released from the cells during spheroplast formation and were therefore isolated in the periplasmic fraction. Of these fractions, which were heterogeneous, it was always the outer membrane subfraction which bound tightly to brush borders. This binding, which was K88 dependent, may have some physiological significance in view of the association between outer membrane fragments and enterotoxin. Thus, released outer membrane fragments equipped with attachment factors may function as enterotoxin carriers which increase the efficiency with which enterotoxin can be delivered to intestinal epithelial cells.

Animals↗
PubMed1984

Rod outer segment membranes: good acceptors for retinoids?

The exchange of all-trans retinoids (retinal, retinol, retinylpalmitate) between PC-vesicles, PC-vesicles and liver microsomes or PC-vesicles and rod outer segment membranes is investigated using 11,12(3)H labeled compounds. In the first two systems, retinal and retinol exchange rapidly, retinyl acetate slowly and retinyl palmitate not at all. Rod outer segment membranes however take up relatively small amounts of retinoids (retinylpalmitate less than retinol less than retinal) and rapidly lose 60-90% of their label in the presence of PC-vesicles. E.G. retinoids clearly favour the PC-vesicle membrane. Apparently, rod outer segment membranes have a much lower affinity for retinoids than other artificial or natural membranes investigated so far.

Animals↗
PubMed1990

Antibody response to Brucella ovis outer membrane proteins in ovine brucellosis.

Hot saline extracts of Brucella ovis were composed of vesicles with outer membrane proteins (OMPs), lipopolysaccharide, and phospholipid as constituents. Extraction with petroleum ether-chloroform-phenol yielded a protein fraction free of detectable lipopolysaccharide, in which group 3 OMPs (28,500 apparent molecular weight [28.5K], 27.0K, and 25.5K) represented 81% of the total. Group 1 OMPs and 67.0K, 22.5K to 21.5K, and 19.5K to 18.0K proteins were also detected. Adsorption of immune sera with whole bacteria suggested that group 3 OMPs and 67.0K, 22.5K to 21.5K, and 19.5K to 18.0K proteins had antigenic determinants exposed on the surfaces of both B. ovis and rough B. melitensis cells but not on smooth B. melitensis cells. Antibodies to group 3 OMPs and the 67.0K protein in the sera of 93 and 87%, respectively, of B. ovis-infected rams were found by immunoblotting. Antibodies to other proteins were present in 67% of these animals. Compared with B. ovis-infected rams which had not developed lesions, rams with epididymo-orchitis had antibodies to a larger variety of proteins. Although ewes infected with B. melitensis also showed antibodies to OMPs, the immunoblot reactions were less intense.

Adsorption↗
PubMed1986

An electron microscope survey of the surface structures and hydrophobicity of oral and non-oral species of the bacterial genus Bacteroides.

Seventeen strains of Bacteroides representing 10 species were examined by negative staining; the majority were from the mouth but a few non-oral strains were included. Seven species had peritrichously-arranged, non-flagellar appendages which could be divided by morphology and ultrastructure into two subgroups, fibrils and fimbriae. Bacteroides asaccharolyticus strains B536 and B537 and Bacteroides gingivalis strains W50, W83, WPH15 and WPH35 had fimbriae with mean width of 4.4 nm and 0.5 to 6.0 microns long depending on the strain. The fimbrial length within each strain also varied. Fibrils were present on two fresh oral isolates of Bacteroides melaninogenicus, Bacteroides intermedius strains T588 and W09, Bacteroides corporis ATCC 33547, Bacteroides oralis ATCC 33269 and Bacteroides buccae ATCC 33574. Fibrils consistently clumped into bundles of variable thickness and formed a fringe around the cell periphery, ranging from 0.27 to 1.2 microns long depending on the strain. Fibril lengths of each strain were uniform. Fibrils had no measurable width and the clumps tapered towards the free ends. Bacteroides loeschii VPI 9085, Bacteroides pentosaceus strains NP333 and J1 and Bacteroides capillosus 29799 had no detectable surface appendages. Fimbriate strains had a layer outside the outer membrane, with a mean thickness of between 17.8 and 28.6 nm. Both fibrillar and fimbriate strains produced many small membranous vesicles budding from the outer membrane. There were two morphological forms of vesicles, ones with either fimbriae or fibrils (species-dependent) and ones with no attached appendages. Of eleven strains tested for cell-surface hydrophobicity by partitioning between hexadecane and buffer, all but one was non-hydrophobic.

Bacteroides↗
PubMed1983

Serotonin-like immunoreactivity and anti-5-hydroxytryptamine (5-HT) antibodies: ultrastructural application in the central nervous system.

Serotonin immunoreactivity of rat neurons was studied using antibodies to 5-hydroxytryptamine (5-HT) raised in rabbits and the peroxidase-antiperoxidase (PAP) technique. The brains were fixed by perfusion with 4% paraformaldehyde + 0.25% glutaraldehyde. Vibratome sections were incubated in 1:1,000 to 25,000 antiserum dilutions. Specificity of 5-HT immunoreactivity was investigated following immunochemical and immunocytochemical criteria. Ultrastructural analysis of the raphe neurons allowed intracellular localization of 5-HT immunoreactivity in somata, dendrites, and synaptic terminals. Granular reaction product deposits were found attached to microtubules, endoplasmic reticulum membranes, synaptic vesicles, and outer mitochondrial membranes. In all cases the reaction product was confined within the limits of the plasma membrane. It is proposed that binding of 5-HT molecules to tissue proteins via a Mannich reaction and formation of intramolecular condensation products (fluorophores) result from aldehyde fixation. The immunogen (5-HT-bovine serum albumin (BSA) complex) exhibits spectrofluorometric emission and excitation maxima similar to those previously described for the 5-HT fluorophore studied in protein films and in fixed tissue. It is concluded that our antibody detects 5-HT-like immunoreactivity preserved in the tissue with the configuration of the fluorophore (i.e., 3,4-dihydrocarboline). Close agreement between the results and previous studies of 5-HT neurons with other techniques (formaldehyde-induced fluorescence, biochemical assays) support the potential value of this antibody.

Animals↗
PubMed1977

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.

Cell Membrane↗
PubMed1995

Rop and Ras2, members of the Sec1 and Ras families, are localized in the outer membranes of labyrinthine channels and vesicles of Drosophila nephrocyte, the Garland cell.

The product of the ras opposite (rop) gene is an essential component of secretion processes in Drosophila. The rop gene product is homologous to the Caenorhabditis elegans UNC-18 and the rat munc-18/n-Sec1/rbSec1 proteins, implicated in the final steps of neurotransmitter exocytosis in nerve terminals, and the bovine mSec1 protein implicated in the secretion of catecholamines in chromaffin cells. The mammalian brain protein has been shown to exert its activity in the presynaptic membrane through transient interaction with syntaxin, an integral component of this membrane. rop is highly expressed in the Drosophila nervous system, where it acts as both a positive and negative modulator of neurotransmitter release. It is also expressed in specialized tissues in which intensive exocytic/endocytic cycles take place, including the garland cells, a small group of nephrocytes which take up waste materials from the hemolymph by endocytosis. rop is regulated by a bidirectional promoter shared with Ras2, a member of the R-ras/TC21 branch of the ras supergene family. Ras2 is also highly expressed in the garland cells. These cells are characterized by their labyrinthine channels, long invaginations extending from the cell membrane, and a rich population of a variety of vesicles. In this study, we analyzed the ultrastructural localization of the Rop and Ras2 proteins in the garland cell. Rop was detected in the outer membranes of the labyrinthine channels, and in the outer membranes of many vesicles located nearby the labyrinthine channels, but not in vesicles located in inner parts of the cell. Using glutathione-S-transferase-syntaxin fusion, we show that Rop is firmly bound to syntaxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Asymmetric orientation of amino groups in the alpha-subunit and the beta-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla.

The orientation of amino groups in the membrane in the alpha- and beta-subunits of (Na+ + K+)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[125I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na+ + K+)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of alpha-subunit, beta-subunit and proteolytic fragments of alpha-subunit. Both the alpha- and the beta-subunit of (Na+ + K+)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the beta-subunit are located on the extracellular surface. In the alpha-subunit, 65-80% of modified groups are localized to the cytoplasmic surface and 20-35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of 125I-labeling within the alpha-subunit. The preservation of (Na+ + K+)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E1- and E2-forms of the alpha-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the beta-subunit being on the extracellular surface, while the alpha-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.

Animals↗
PubMed2003

Modulation of Porphyromonas gingivalis proteinase activity by suboptimal doses of antimicrobial agents.

BACKGROUND: Antimicrobial agents are sometimes used as adjuncts for the treatment of aggressive and refractory forms of periodontitis. In this study, we used a culture plate assay to investigate the effect of suboptimal doses of antimicrobial agents on proteinase activity of Porphyromonas gingivalis. METHODS: A culture plate assay using gelatin as the substrate, which allows a semiquantitative determination of proteinase activity, was developed. Suboptimal inhibitory concentrations of tetracycline, minocycline, doxycycline, metronidazole, penicillin G, or chlorhexidine were added to the medium, and proteolysis zones were determined following the growth of three strains of P. gingivalis. The effect of antimicrobials on outer membrane vesicle-associated gingipains also was determined. RESULTS: The gelatin plate assay was a convenient, simple procedure for investigating the effect of suboptimal inhibitory concentrations of antimicrobial agents on proteinases produced by P. gingivalis. The largest reduction (> 75%) in the proteolysis zones produced by three strains of P. gingivalis was obtained with minocycline. Tetracycline and doxycycline also reduced the proteolysis zones. A suboptimal inhibitory concentration of chlorhexidine increased the proteolysis zones by up to 70%. Metronidazole and penicillin G produced no noticeable effect. The suboptimal inhibitory concentrations of minocycline, tetracycline, and doxycyline did not reduce the activity of outer membrane vesicle-associated Arg- and Lys-gingipains. CONCLUSION: Results from this study suggest that sublethal concentrations of some antimicrobial agents in subgingival sites have the potential to affect the physiology of P. gingivalis, notably by increasing or decreasing the proteolytic activity of the bacteria.

Adhesins, Bacterial↗
PubMed1995

Surface charge, fluidity, and calcium uptake by rat intestinal brush-border vesicles.

Biological membrane outer surfaces are negatively charged and interact with positively charged calcium ion during calcium uptake. Positively charged polycations such as polyarginine bind to membranes with high affinity, displacing bound calcium from the membrane. We tested the effect of polyarginine on uptake of calcium by brush-border membrane vesicles and examined the responses in terms of membrane fluidity by electron paramagnetic resonance (EPR). Polyarginine inhibited the saturable component of calcium uptake by a mechanism combining inhibition characteristics of strontium (competitive) and magnesium (non-competitive). Unlike the inhibition of non-saturable calcium uptake by strontium and magnesium, polyarginine increased kD, the rate constant for non-saturable calcium uptake, by a concentration dependent mechanism. These effects of polyarginine on calcium uptake were associated with decreased membrane fluidity at the uptake temperature. These findings are consistent with a role for surface negative charge in determining both saturable and non-saturable calcium uptake. Increased membrane fluidity is associated with decreased saturable and increased non-saturable calcium uptake. Although increased fluidity might be involved in the increased kD for non-saturable uptake, the concentration-specific stimulating effect of polyarginine suggests a gating mechanism.

Animals↗
PubMed1989

Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane.

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.

Antibodies↗
PubMed1992

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.

Animals↗