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 451 records · Page 25Linked to original sources
PubMed2004

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles.

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; rho=1.18 g cm(-3)) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Amino Acid Sequence↗
PubMed1991

Secretion of the cell surface lipoprotein pullulanase in Escherichia coli. Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway.

The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion. When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles. In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis. Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes. A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled. We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.

Base Sequence↗
PubMed1989

pH gradient-stimulated phosphate transport in outer medullary brush-border membranes.

Phosphate transport was studied in brush-border membrane vesicles prepared from outer medullary tissue of the porcine kidney. Phosphate uptake studies were performed in the absence of sodium at 21 degrees C. A 1.2- to 12-fold overshoot, above equilibrium values, was present with intracellular pH (pHin) equal to 8.0 and extracellular pH (pHout) equal to 6.5, which was not evident at pHin = pHout. Concentration-dependence of the pH-stimulate uptake was determined by the difference of uptake in the absence of a pH gradient (pHin = pHout) from that in the presence of a pH gradient over a large range of phosphate concentrations. The uptake was consistent with a single facilitative system characterized by apparent kinetic parameters; with Michaelis constant 149 +/- 11 microM and maximal velocity 4.9 +/- 0.4 nmol.mg protein-1.min-1, n = 3. Phosphate uptake was inhibited by the stilbene derivative 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid with a mean inhibition constant (Ki) value of 0.15 mM (n = 2). In addition, pH gradient-stimulated phosphate uptake was sensitive to furosemide and bumetanide; Ki values of 0.50 +/- 0.05 and 0.11 +/- 0.04 mM, respectively. Arsenate (1 mM) and phosphonoformate (1 mM) inhibited pH-dependent phosphate uptake, whereas sulfate (5 mM), bicarbonate (25 mM), and chloride (100 mM) were without effect, indicating that the transport system is relatively specific to phosphate and its close analogues. pH gradient-stimulated phosphate uptake was not influenced by potassium-diffusional gradients. The data provide evidence for a facilitative process in brush-border membrane vesicles isolated from outer medullary tissue of the pig kidney that is capable of transporting phosphate in the absence of sodium.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Double-layered membrane vesicles released from mammalian cells infected with Sendai virus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-M(VSV)). When cells were infected with SeV-M(VSV), VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (>/= 0.8 microm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M.

Animals↗
PubMed1992

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.

Animals↗
PubMed2002

Virus-assisted loading of polymer nanocontainer.

We present a DNA-containing polymeric nanocontainer using the self-assembled superstructure of amphiphilic block copolymers in aqueous solutions. To demonstrate that DNA translocation is possible across a completely synthetic block copolymer membrane, we have used a phage transfection strategy as a DNA-transfer model system. For this purpose the bacterial channel forming protein LamB was reconstituted in ABA-triblock copolymer vesicles. The outer membrane protein LamB is a specific transporter for maltodextrins but also serves as a receptor for lambda phage to trigger the ejection of lambda phage DNA. We demonstrate that the functionality of the LamB protein is fully preserved despite the artificial surrounding. This leads to a type of polymeric vehicle for DNA that could be useful for gene therapy.

Bacterial Outer Membrane Proteins↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗
PubMed2003

Endosomal compartmentalization in three dimensions: implications for membrane fusion.

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.

Animals↗
PubMed2026

Genome-guided isolation and characterization of a novel bacteriophage infecting Escherichia coli reveal a putative new genus.

We have isolated and characterized a novel bacteriophage termed Jab, with lytic activity against multidrug-resistant clinical isolates of Escherichia coli. Phage Jab was identified from liquid manure by means of metagenome sequencing of a phage community enrichment using an E. coli clinical isolate ECH07 as host. The initial enrichment was composed of four phages, of which phage Jab represented only a minute fraction (less than 1%). Jab isolation strategy comprised a targeted approach using iterative replication rounds while equipping ECH07 with resistance against the numerically dominant phages coupled with a subsequent host switch to E. coli BL21. Whole-genome sequence analysis revealed only a remote evolutionary distance to known phages within the subfamily Vequintavirinae. The dsDNA genome of phage Jab comprises 142,100 bp (GC content 40.09%) and encodes 264 proteins and five transfer RNAs (tRNAs). No lysogeny-associated proteins were detected, suggesting an obligate lytic lifestyle. In silico genome analysis revealed the presence of at least four putative depolymerases. The closest homology of phage Jab is with members of the new genus Septuagintavirus with around 34% nucleotide identity. VIRIDIC and network analyses strongly suggest that phage Jab belongs to a putative novel genus. The host range of phage Jab is likely restricted to E. coli, displaying a moderately narrow host range (i.e., productive lysis in 8 out of 27 isolates tested). Notably, transmission electron microscopy (TEM) revealed the occurrence of conspicuous unique spherical structures attached at the end of the tail fibers when propagated on BL21 but not when propagated on ECH07. Although their function remains enigmatic, the possible role of those structures as a bacterial (vesicle-based) defense mechanism warrants further investigation.

Escherichia coli↗
PubMed1994

Interaction of calmodulin with the cyclic GMP-gated channel of rod photoreceptor cells. Modulation of activity, affinity purification, and localization.

The cGMP-gated cation channel of rod photoreceptor cells plays a central role in the phototransduction process by controlling the influx of cations into the rod outer segment in response to changes in cGMP levels. Previous studies have shown that the cGMP-gated channel in native rod outer segment membrane vesicles is modulated by calmodulin in a calcium-dependent manner. In this study we report that the immunoaffinity-purified channel consisting of the 63-kDa alpha-subunit and a 240-kDa protein is also modulated by calmodulin when reconstituted into lipid vesicles. In the absence of calmodulin, the purified channel had an apparent Km of 33 microM and a Hill coefficient of 3.3 for cGMP-dependent efflux of Ca2+ from reconstituted lipid vesicles. In the presence of calmodulin, the Km increased to 44 microM without affecting the Hill coefficient or maximum velocity of ion efflux. Calmodulin modulation of the channel is inhibited by the calmodulin antagonist, mastoparan. In the absence of mastoparan, the half-maximum inhibition of channel activity (IC50) occurred at 1.85 +/- 0.25 nM calmodulin at a cGMP concentration of 12.5 microM; in the presence of mastoparan, the IC50 value increased to 20.3 +/- 3.8 nM calmodulin. Based on the strong, selective interaction of calmodulin with the channel, an efficient, general method has been developed to isolate functionally active cGMP-gated channels from mammalian and amphibian photoreceptor membranes. Calmodulin extraction studies, Western blotting, and channel activity measurements indicate that endogenous rod outer segment calmodulin modulates the activity of the channel through its binding to the 240-kDa protein. From these studies we conclude that the 240-kDa protein of the cGMP-gated channel is a major calmodulin target protein of rod outer segment membranes.

Animals↗
PubMed2005

Quantification and distribution of big conductance Ca2+-activated K+ channels in kidney epithelia.

Big conductance Ca2+ activated K+ channels (BK channels) is an abundant channel present in almost all kind of tissue. The accurate quantity and especially the precise distribution of this channel in kidney epithelia are, however, still debated. The aim of the present study has therefore been to examine the presence of BK channels in kidney epithelia and determine the actual number and distribution of these channels. For this purpose, a selective peptidyl ligand for BK channels called iberiotoxin or the radiolabeled double mutant analog 125I-IbTX-D19Y/Y36F has been employed. The presence of BK channels were determined by a isotope flux assay where up to 44% of the total K+ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies, 125I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex, outer medulla and inner medulla with Bmax values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography and immunohistochemical studies. In cortical collecting ducts, BK channels were exclusively located in principal cells while no channels could be found in intercalated cells. The abundant and distinct distribution in kidney epithelia talks in favor for BK channels being important contributors in maintaining salt and water homeostasis.

Animals↗
PubMed1994

Na(+)-Ca2+,K+ exchange in bovine retinal rod outer segments: quantitative characterization of normal and reversed mode.

Ca2+ homeostasis of bovine retinal rod outer segments is maintained through Na(+)-Ca2+,K+ exchangers and cGMP-gated channels in the plasma membrane. It has recently been demonstrated that both proteins are associated. This novel finding allowed us to investigate quantitatively normal and reversed mode Na(+)-Ca2+,K+ exchange in rod outer segment membrane vesicles and reconstituted proteoliposomes both containing exchangers in rightside-out and inside-out orientations. Addition of Na+ activated both normal and reversed mode exchange; if, however, initially Ca2+ from vesicles containing inside-out oriented exchangers has been released by activation of the associated channels, only normal mode exchange was observed upon addition of Na+. Using this approach, the fractions of vesicles containing rightside-out and inside-out oriented exchangers were about similar in these vesicle preparations. Normal and reversed mode exchange had similar Na+ concentrations of about 70 mM for half maximal activation (in the presence of 115 mM K+) and cooperativity parameters, nHill, of about 2.0. Furthermore, both modes were electrogenic, and showed only little Na(+)-Ca2+,K+ exchange in the absence of K+. The two modes of exchange differed, however, in the maximal exchange rate, the normal mode being about twice as fast as the reversed mode.

Animals↗
PubMed1997

Lipids may not be involved in the recognition of apocytochrome C during its transportation.

It was shown that apocytochrome c was less insertion into monolayers from outer mitochondrial membrane lipids than into those from microsomal membrane lipids; the alpha-helix content of apocytochrome c induced by small unilamellar vesicles prepared from outer mitochondrial membrane lipids was less than by those from microsomal membrane lipids; the import efficiency of apocytochrome c into large unilamellar vesicles from outer mitochondrial membrane lipids was also lower than into those from microsome membrane lipids. No specific affinity between apocytochrome c and outer mitochondrial membrane lipids could be found. Import of apocytochrome c across the intact mitochondria, sealed outer mitochondrial membrane and microsome membrane vesicles was compared. Results showed that apocytochrome c was accumulated only in mitochondria, but not the other two kinds of vesicles.

Animals↗
PubMed2001

Three distinct lipid kinase activities are present in spinach chloroplast envelope membranes: phosphatidylinositol phosphorylation is sensitive to wortmannin and not dependent on chloroplast ATP.

Chloroplast envelope membranes display properties that are important in lipid synthesis, regulation of metabolites, and protein transport, as well as in signal transduction. The recent discovery showing that phosphorylation of lipids occurs in envelope membranes provides a new approach for understanding the role of chloroplast lipids in these processes. The present investigation shows that three major lipid kinase activities are at least present in envelope membranes. These activities greatly depend on external conditions, such as pH, ATP concentrations, temperature, and chloroplast ATP and wortmannin sensitivity. Two types of phosphorylated lipid couples displayed similar intrinsic responses toward these biochemical parameters, namely phosphatidic acid (PA) and its lysoderivative (LPA) and monogalactosyl-phosphate-diacylglycerol (MGpDG) and its lysoderivative (LMGpDG), but not phosphatidylinositol-monophosphate (PIP) and its lysoderivative (LPIP). Phosphorylation of phosphatidylinositol was not dependent on chloroplast ATP, but was sensitive toward wortmannin in intact chloroplasts and outer envelope membrane vesicles.

Adenosine Triphosphate↗
PubMed1988

1,25-Dihydroxyvitamin D stimulates sodium-dependent phosphate transport by renal outer cortical brush-border membrane vesicles by directly affecting membrane fluidity.

We examined the effects of several forms of vitamin D added to renal brush-border membrane suspensions on phosphate and glucose transport and on membrane fluidity. The 1,25-D stimulated and the other vitamin D decreased phosphate uptake. In contrast, glucose uptake was not affected by the treatment of vitamin D. The 1,25-D resulted in a significant shift of the lower transition temperature in Arrhenius plots for phosphate, but not for glucose uptakes, from 15 degrees C to 11.5 degrees C. These data indicate that the 1,25-D may alter membrane fluidity, limited to the phosphate transporter, thus affecting the phosphate uptake.

Animals↗
PubMed1986

Immunoelectronmicroscopic localization of actin in ionophore-treated boar sperm.

Actin in ionophore-A23187 treated boar sperm has been localized by indirect immunofluorescence and immunoelectronmicroscopy (IEM), using an anti-actin monoclonal antibody. By IEM, after en bloc staining technique and treatment with 15 nm colloidal gold-IgM complex, actin was found associated with the plasma membrane (PM) and the outer acrosomal membrane vesicles and under the PM of the equatorial segment.

Acrosome↗
PubMed1994

Reproduction of bovine keratoconjunctivitis with a purified haemolytic and cytotoxic fraction of Moraxella bovis.

Evidence that the beta-haemolysin produced in vitro by Moraxella bovis is an important virulence determinant in vivo was strengthened by studies using a haemolytic preparation of greater purity than previously available. A concentrated haemolytic fraction containing outer-membrane bound vesicles was separated from the cell-free filtrate of a bacterial culture using a process comprising tangential flow ultrafiltration, ion-exchange and gel-filtration high-performance liquid chromatography and centrifugal-driven filtration. The cytotoxicity of haemolytic fractions for calf-corneal epithelial cells in vitro was investigated at progressive stages of this attempted haemolysin purification procedure and the results demonstrated a positive correlation for the levels of haemolytic and cytotoxic activity throughout. Further support for the role of the haemolysin was obtained in vivo following the intra-corneal injection of calves with a crude or a purified haemolytic fraction. The ocular damage caused by both preparations, together with the healing processes and microscopic pathology of the experimentally induced damage closely resembled published descriptions of naturally occurring infectious bovine keratoconjunctivitis. No effect was obtained in vitro or in vivo from equivalent fractions prepared from a non-haemolytic strain of M. bovis.

Animals↗