PubMed Health⌕ Search

Biomedical subjects

W D Niles

Publications and source records attributed to W D Niles.

26 records · Page 2Linked to original sources
PubMed1989

Hydrostatic pressures developed by osmotically swelling vesicles bound to planar membranes.

When phospholipid vesicles bound to a planar membrane are osmotically swollen, they develop a hydrostatic pressure (delta P) and fuse with the membrane. We have calculated the steady-state delta P, from the equations of irreversible thermodynamics governing water and solute flows, for two general methods of osmotic swelling. In the first method, vesicles are swollen by adding a solute to the vesicle-containing compartment to make it hyperosmotic. delta P is determined by the vesicle membrane's permeabilities to solute and water. If the vesicle membrane is devoid of open channels, then delta P is zero. When the vesicle membrane contains open channels, then delta P peaks at a channel density unique to the solute permeability properties of both the channel and the membrane. The solute enters the vesicle through the channels but leaks out through the region of vesicle-planar membrane contact. delta P is largest for channels having high permeabilities to the solute and for solutes with low membrane permeabilities in the contact region. The model predicts the following order of solutes producing pressures of decreasing magnitude: KCl greater than urea greater than formamide greater than or equal to ethylene glycol. Differences between osmoticants quantitatively depend on the solute permeability of the channel and the density of channels in the vesicle membrane. The order of effectiveness is the same as that experimentally observed for solutes promoting fusion. Therefore, delta P drives fusion. When channels with small permeabilities are used, coupling between solute and water flows within the channel has a significant effect on delta P. In the second method, an impermeant solute bathing the vesicles is isosmotically replaced by a solute which permeates the channels in the vesicle membrane. delta P resulting from this method is much less sensitive to the permeabilities of the channel and membrane to the solute. delta P approaches the theoretical limit set by the concentration of the impermeant solute.

Hydrostatic Pressure↗
PubMed1988

Planar bilayer membranes made from phospholipid monolayers form by a thinning process.

We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.

Electric Conductivity↗
PubMed1987

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents.

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.

Adsorption↗
PubMed1985

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.

Fluoresceins↗
PubMed1985

Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.

Bacterial Outer Membrane Proteins↗
PubMed1982

Effects of hypertonic solutions on quantal transmitter release at the crayfish neuromuscular junction.

1. The effects of a hypertonic bathing medium, containing either NaCl or melezitose, on the average number (m) and the time course of quantal release following an action potential were studied using focal extracellular recording methods at synaptic sites on the opener muscle of the crayfish leg.2. After the application of hypertonic saline, the rate of spontaneous quantal release increased but m decreased to a new level within 1 min; the extent of depression depended on the magnitude of the increase in tonicity until the osmolarity was 50% greater than the normal value of 0.43 osmol/l (Osm), but greater increases in tonicity exerted little further effect.3. The synaptic delays were increased and distributed over a longer range of time in hypertonic solutions; also, the latency between the first and second quantal releases in a multiple response to a single nerve impulse was also increased.4. Hypertonicity had no significant effect on the conduction velocity of the action potential, the independence of successive quantal releases in the same response, or the uniformity of the rise and fall of the probability of quanta release following an action potential, alpha(t).5. The time course of alpha(t) is prolonged in hypertonic solutions; this was manifested as an increase in the time constant of the exponential decline in alpha(t) from its peak value following the nerve impulse.5. When the potentiating agent 5-hydroxytryptamine (5-HT) was added to the hypertonic saline, m was increased, and the time course of alpha(t) was prolonged further than in the hypertonic solution alone; 5-HT produced no change in the time course of alpha(t) when it was added to normal saline, although m was increased.6. It is concluded that in this preparation hypertonicity decreases the rate of release of each quantum from the nerve terminal following a nerve impulse.

Action Potentials↗
PubMed1982

Anion blockers inhibit impulse-evoked quantal transmitter release at the crayfish neuromuscular junction.

1. The effects of the anion-transport blocking reagents Probenecid, 4,4'diisothiocyanostilbene-2,2'-disulphonate (DIDS), and pyridoxal phosphate on the number of quanta released (m) and the time course of the probability of quantal release, alpha(t), following an action potential were studied at single synaptic sites on the opener muscle in the crayfish leg.2. Low concentrations (10-50 muM) of DIDS or Probenecid produced decreases in m to about 30-50% of the initial value; at higher concentrations (100 muM or greater), evoked release failed. Much greater concentrations (500 muM) of pyridoxal phosphate were necessary to produce decreases in m.3. Synaptic depression in 50 muM-Probenecid was reversible by removing the agent; after treatment with 50 muM-DIDS, however, m partially regained the initial value upon removal of the drug.4. The time course of alpha(t) after an action potential was unaffected by any of the reagents.5. It is concluded that the reagents decrease the number of quanta evoked by a nerve impulse by decreasing the magnitude, but not the time course, of the average rate of quantal release following an action potential; it is suggested that nerves and other secretory cells may share a common mechanism during exocytosis that is subject to modification by these agents.

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