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At least 685 records · Page 38Linked to original sources
PubMed1976

Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strain of Salmonella.

The effect of polymyxin on two sets of Salmonella mutants was studied by thin-section and scanning electron microscopy. Polymyxin (in increasing concentrations, starting just below bactericidal effect) caused the appearance of the previously described rodlike projections on the cell surface of wild-type (smooth, polymyxin-sensitive) bacteria. These projections seemed to involve the outer membrane of the cell wall. In rough mutants, which are deficient in lipopolysaccharide, the projections were much smaller and flat. Higher concentrations of polymyxin were required to produce morphological effects in polyxmin-resistant mutants of both smooth and rough forms. Furthermore, in these mutants polymyxin caused vesicle-like bulging of the total outer membrane quite different in appearance from the rodlike projections of the wild type.

Cell Membrane↗
PubMed1995

Transit sequence-dependent binding of the chloroplast precursor protein ferredoxin to lipid vesicles and its implications for membrane stability.

The binding of the transit peptide (trfd) and precursor of the chloroplast protein ferredoxin (prefd) to large unilamellar lipid vesicles was investigated in relation to the lipid composition of the bilayer. Prefd binds with a dissociation constant of 0.27 microM to vesicles with a composition corresponding to the chloroplast envelope outer membrane. Binding is mediated by the transit sequence. From an analysis of binding to vesicles containing the individual lipid components it could be concluded that anionic lipids are mainly responsible for binding, emphasizing the importance of electrostatics for the transit sequence-lipid interaction. Binding is also mediated by the specific chloroplast glycolipid monogalactosyldiacylglycerol. Monolayer experiments revealed that in this case a more extended domain of the transit sequence inserts into the lipid layer. Precursor binding does not result in a loss of vesicle barrier function. However, high concentrations of trfd do cause release of vesicle-enclosed carboxyfluorescein. The results are discussed in the light of the chloroplast protein import process, with special emphasis on the role of monogalactosyldiacylglycerol.

Amino Acid Sequence↗
PubMed1996

Cyclic GMP-gated channels of bovine rod photoreceptors: affinity, density and stoichiometry of Ca(2+)-calmodulin binding sites.

1. Ca(2+)-loaded vesicles of bovine rod outer segment (ROS) membranes were used to examine the influence of Ca(2+)-calmodulin (Ca(2+)-CaM) on the activity of cGMP-gated channels. 2. In vesicles prepared from ROS membranes which were washed at zero free Ca2+, Ca(2+)-CaM reduced the Ca2+ flux to maximally 40%. The dose-response curve for activation of the cGMP-gated channel had a half-maximal value of 36.8 +/- 2 microM in the CaM-free state, and of 55.6 +/- 5.2 microM in the Ca(2+)-CaM-bound state. In both cases the Hill coefficients were 2.2 +/- 0.2. 3. In vesicles prepared from ROS membranes which were washed at 100 microM Ca2+, the dose-response curve was identical to the Ca(2+)-CaM-bound state. 4. Titration of the Ca(2+)-CaM-dependent decrease of the channel activity upon addition of 40 microM cGMP yielded half-maximal Ca(2+)-CaM concentrations (EC50CaM) which were linearly correlated with the concentration of membrane vesicles. Extrapolation of EC50CaM to infinite dilution of vesicles yielded a Ca(2+)-CaM affinity constant for the cGMP-gated channel of 1.01 +/- 0.20 nM. Hill analysis of the Ca(2+)-CaM titrations resulted in a Hill coefficient of 1.36 +/- 0.15. 5. From the slope of the linear regression of EC50CaM plotted vs. the rhodopsin concentration, the molar ratio of rhodopsin to externally accessible Ca(2+)-CaM binding sites of fused ROS membranes was determined to be 1439 +/- 109. Therefore, there are about 720 molecules of rhodopsin per Ca(2+)-CaM binding site present in ROS. 6. Based on these data, a density of 560 Ca(2+)-CaM binding sites micron-2 is estimated for the plasma membrane of bovine ROS, suggesting that there are two Ca(2+)-CaM binding sites per channel. 7. The Ca(2+)-CaM effect did not become noticeable until the ROS membranes were hypotonically washed at free [Ca2+] below 100 nM, suggesting that an endogenous Ca(2+)-binding protein was washed off in the absence of Ca2+. 8. If the endogenous Ca(2+)-binding protein of bovine ROS membranes was washed off at zero Ca2+ and then Ca(2+)-CaM added, Ca(2+)-CaM could only be washed off again at free [Ca2+] below 100 nM. 9. These findings strongly suggest that the endogenous Ca(2+)-binding protein of the bovine cGMP-gated channel is CaM.

Animals↗
PubMed1987

Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force.

In order to reach their final destination, periplasmic and outer membrane proteins have to pass the cytoplasmic membrane of Escherichia coli cells. To study the transport of PhoE protein, we developed an in vitro transcription-translation and translocation system. In this in vitro system, the protein is synthesized as a larger precursor, which can be processed by purified leader peptidase. The precursor can be translocated into inverted inner membrane vesicles as judged by the protection against externally added protease. Only part of the translocated protein is in the processed mature form. Translocation can occur posttranslationally and requires both ATP and the protonmotive force for an optimal process. Upon incubation of vesicles with mature PhoE protein or precursor PhoE in the absence of ATP, the proteins are bound to the vesicles, but they are not translocated, since they are still sensitive to externally added protease.

Adenosine Triphosphate↗
PubMed1979

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.

Cell Membrane↗
PubMed1980

Topological studies of the membrane-binding segment of cytochrome b5 embedded in phosphatidylcholine vesicles.

Carboxypeptidase Y (CPY) digestion of cytochrome b5 incorporated into single-walled liposomes of egg phosphatidylcholine (PC) resulted in the release of 25 amino acid residues form each molecule of the cytochrome and concomitant detachment of the cytochrome from the vesicles. This finding suggests that the COOH-terminus of the incorporated cytochrome is exposed to the outer surface of the liposomal membrane, since the cytochrome-carrying vesicles were shown to be impermeable to macromolecules such as dextran T-40 by isopycnic centrifugation studies. The possibility that CPY had attacked a small amount of free cytochrome b5 which was in equilibrium with the liposome-bound cytochrome could be excluded by the finding that transfer of the bound cytochrome to the unbound state was a much slower process than CPY-induced detachment of cytochrome b5. CPY could also attack the COOH-terminus of cytochrome b5 embedded in dipalmitoyl-PC liposomes even below the phase transition temperature of the synthetic phospholipid. Moreover, the tyrosine residue(s) near the COOH-terminus of the PC vesicle-bound cytochrome could be readily iodinated by the action of lactoperoxidase added externally. These observations indicate that the COOH-terminus of the liposome-bound cytochrome, like its heme-containing, hydrophilic domain, is exposed to the outer surface of the vesicular membrane. The cytochrome detached from the vesicles by CPY digestion could neither rebind to the liposomes nor form an oligomeric aggregate, suggesting that CPY had removed all the amino acid residues which are required for the protein to bind to membranes. The mechanism of CPY-induced detachment of cytochrome b5 from liposomes and the size of the peptide segment which is directly involved in the interaction of the cytochrome with the lipid bilayer membranes are discussed.

Amino Acid Sequence↗
PubMed2003

Matrix vesicles and calcification.

Matrix vesicles (MVs) are extracellular, 100 nM in diameter, membrane-invested particles selectively located at sites of initial calcification in cartilage, bone, and predentin. The first crystals of apatitic bone mineral are formed within MVs close to the inner surfaces of their investing membranes. Matrix vesicle biogenesis occurs by polarized budding and pinching-off of vesicles from specific regions of the outer plasma membranes of differentiating growth plate chondrocytes, osteoblasts, and odontoblasts. Polarized release of MVs into selected areas of developing matrix determines the nonrandom distribution of calcification. Initiation of the first mineral crystals, within MVs (phase 1), is augmented by the activity of MV phosphatases (eg, alkaline phosphatase, adenosine triphosphatase and pyrophosphatase) plus calcium-binding molecules (eg, annexin I and phosphatidyl serine), all of which are concentrated in or near the MV membrane. Phase 2 of biologic mineralization begins with crystal release through the MV membrane, exposing preformed hydroxyapatite crystals to the extracellular fluid. The extracellular fluid normally contains sufficient Ca2+ and PO4(3-) to support continuous crystal proliferation, with preformed crystals serving as nuclei (templates) for the formation of new crystals by a process of homologous nucleation. In diseases such as osteoarthritis, crystal deposition arthritis, and atherosclerosis, MVs initiate pathologic calcification, which, in turn, augments disease progression.

Bone Matrix↗
PubMed1993

[Ultrastructure and properties of Actinobacillus actinomycetemcomitans].

In this study, plaque samples taken from periodontal pockets were cultured on Tryptic-soy-serum-bacitracin-vancomycin agar and identified according to colony morphology, Gram stain, catalase reaction and nitrate reduction tests after an incubation of 48 hours in candle jar. Cells identified as Actinobacillus actinomycetemcomitans were evaluated under the transmission electron microscope for fine structure. Outer membrane was observed as an irregular structure containing microcapsules. Unlike previous studies, periplasmic space was observed separately from other layers. Cytoplasmic membrane was observed surrounded by an electron-opaque cytoplasm. Besides, extracellular vesicles were seen extending to the outer environment around cytoplasmic membrane. Pathogenic properties of organelles were discussed. As a result, it was concluded that, in the future studies, products released and structural alterations occurring during growth of the organism, their pathologic effects and ways to suppress them need to be investigated microbiologically, ultrastructurally and biochemically.

Actinobacillus Infections↗
PubMed1995

Ultrastructural localization of SNAP-25 within the rat spinal cord and peripheral nervous system.

Synaptosomal associated protein of 25 kDa (SNAP-25) has been implicated in the membrane fusion machinery of neurotransmitter release and axonal growth. Using immunocytochemistry, we have analyzed the distribution and ultrastructural localization of SNAP-25 in selected areas of the central and peripheral nervous systems of adult rats. We show that the protein is specifically expressed in the trans face of the Golgi apparatus and in the axonal compartment. In axons and nerve endings, SNAP-25 is localized to discrete areas of the membranes of most organelles such as the axoplasmic reticulum, the axolemma, the outer membrane of mitochondria and synaptic vesicles. This wide distribution of SNAP-25 suggests that the protein is involved in the fusion of membranes in the whole axonal compartment of neurons.

Animals↗
PubMed1992

Gating of retinal rod cation channel by different nucleotides: comparative study of unitary currents.

Single channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K0.5 is about 3 microM for 8Br-cGMP, 18 microM for cGMP and 740 microM for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni2+ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMP- or 8Br-cGMP-dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry 28:2785-2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases Po; addition of cAMP concentrations higher than about 70 microM progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol. 123:133-147).

Animals↗
PubMed1992

Localization of malachite green positive lipids in the matrix of bone nodule formed in vitro.

An electron histochemical study was carried out on bone nodules formed in vitro in collagenase-released calvarial cells in order to visualize the lipid components of the extracellular matrix (EM). The malachite green aldehyde fixative technique, which allows both preservation and staining of some phospholipids of the extracellular matrix, was used. Controls were performed on sections demineralized, and then submitted to lipid extraction with a chloroformmethanol mixture (2/1 v/v) and to glycosaminoglycans digestion with 0.5% bovine testicular hyaluronidase to verify specificity for lipid staining. This allowed us to visualize the lipids (1) in the osteoid as granules associated to ribbon-like structures connected to the collagen fibers, (2) as electrondense deposits seen as dots on the outer surface membrane of the matrix vesicles, and (3) in the mineralized matrix as roundish patches formed of needle-shaped materials and at the mineralization front as individual ones. This study demonstrated that at the EM level, the lipids are present in the osteoid at locations very similar to what have been observed for the glycosaminoglycans, and in the mineralized matrix as components of the crystal ghosts.

Animals↗
PubMed1978

[Electron microscopic and morphometric investigations of the integument of Acanthocephala (Aschelminthes) (author's transl)].

The integument of the acanthocephalans Echinorhynchus gadi, Acanthocephalus lucii, Polymorphus minutus and Macracanthorhynchus hirudinaceus have been investigated by stereoscan and transmission electron microscopy. The absorptive surface is considerably increased by invaginations of the outer plasma membrane (pore canals and vesicles). The rate of this enlargement has been calculated using morphometric methods. The values range from 20 to 62. The values are compared to values obtained from other gutless parasitic helminths and from the free-living rotifers which are presumably to be regarded as closely related to the acanthocephalans.

Acanthocephala↗
PubMed1990

Cl- transport in basolateral renal medullary vesicles: I. Cl- transport in intact vesicles.

This paper provides the results of studies which characterized conductive 36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive 36Cl- uptake was studied under two different experimental conditions. In the first, 36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive 36Cl- uptake. By these two methods, voltage-sensitive 36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total 36Cl- flux. Separate paired studies demonstrated that the conductive 36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 microM. The voltage-dependent 36Cl- uptake had an activation energy of 6.4 kcal/mole. This 36Cl- conductance had an anion selectivity sequence of I- greater than Cl- greater than or equal to NO3- much greater than gluconate.

Animals↗
PubMed1988

Taurine transport by rabbit kidney brush-border membranes: coupling to sodium, chloride, and the membrane potential.

Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na+-D-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na+ gradient (K+ diffusion potential) and could be completely inhibited by other beta-amino acids. It displayed a specific anion requirement (Cl- greater than or equal to Br- much greater than SCN- greater than I- greater than NO-3). At chemical Na+ equilibrium, Cl- gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl- gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl- as well as Br-. The Cl- stoichiometry was found to be one, whereas taurine transport, in the presence of Cl-, was sigmoidally related to the Na+ concentration, resulting in a coupling ratio of 2 to 3 Na+: 1 taurine. Upon Cl- replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na+ concentration (up to 150 mM). These results suggest a 2 to 3 Na+ :1 Cl- :1 taurine cotransport mechanism driven mainly by the Na+ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl- appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.

Animals↗
PubMed1991

Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers.

Unitary currents through cGMP-dependent channels of retinal rods are observed following incorporation into planar lipid bilayers of native vesicles from purified rod outer segment membranes washed free of soluble and peripheral proteins. The influence of the concentration of cGMP, inhibitors (cis-diltiazem, tetracaine and Ag+) and divalent cations (Ca2+, Mg2+, and Co2+) on the conductance and open probability of the channel is described, as well as the voltage dependence of these effects. The cGMP dependence suggests the existence of four binding sites for cGMP and reveals that sequential binding of four cGMP molecules corresponds to the opening of four discrete conductance levels. Finally, we provide conclusive evidence that activated G-protein does not directly inactivate the cGMP-dependent channels of bovine retinal rods.

Animals↗
PubMed1992

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.

Affinity Labels↗
PubMed1990

Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode.

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.

Animals↗
PubMed1997

Sub-mitochondrial localization of the catalytic subunit of pyruvate dehydrogenase phosphatase.

Using a specific antibody against the PDP catalytic subunit, PDPc, precise localization of this subunit in mitochondria was performed. Sub-fractionation of purified mitochondria by controlled swelling processes led to the isolation of outer membranes, matrix space and inner membrane vesicles which were purified on a sucrose density gradient. In this study, we demonstrated that PDPc was not recovered as a soluble protein in the matrix space but was associated with the inner membrane. Moreover, Triton X-114 phase partitioning performed on inner membranes showed that PDPc behaved both as a hydrophilic and as a hydrophobic protein, thus suggesting two different forms of this enzyme.

Amino Acid Sequence↗