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F R Hallett

Publications and source records attributed to F R Hallett.

At least 19 recordsLinked to original sources
PubMed2001

Osmotically induced shape changes of large unilamellar vesicles measured by dynamic light scattering.

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.

Elasticity↗
PubMed2000

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids.

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.

Buffers↗
PubMed2000

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes.

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.

Animals↗
PubMed2000

Small-angle neutron scattering from large unilamellar vesicles: an improved method for membrane thickness determination.

Small-angle neutron scattering (SANS) measurements were performed on large unilamellar vesicles (LUVs) in order to investigate solute effects on membrane properties. Although SANS is a well established technique for the measurement of membrane thickness in unilamellar vesicles, earlier measurements have depended on approximate treatments of the scattering function and have suffered from effects of multilamellarity or difficulty in sample preparation. More recent studies of temperature induced thickness changes in DPPC LUVs which have included explicit treatment of the full scattering function were complicated by disparities between the predicted and measured scattering curves. Here, we reexamine theoretical descriptions of SANS from LUVs. Motivated by our observations, we then introduce a new method for interpretation of SANS data, which we compare to established techniques and apply to our measurements.

Lipid Bilayers↗
PubMed1999

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.

Bacterial Proteins↗
PubMed1996

Optical changes in unilamellar vesicles experiencing osmotic stress.

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.

Lipid Bilayers↗
PubMed1995

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.

Biophysical Phenomena↗
PubMed1993

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.

Biomechanical Phenomena↗
PubMed1993

Mechanical properties of vesicles. II. A model for osmotic swelling and lysis.

Vesicle polydispersity and leakage of solutes from the vesicle lumen influence the measurement and analysis of osmotically induced vesicle swelling and lysis, but their effects have not been considered in previous studies of these processes. In this study, a model is developed which expressly includes polydispersity and leakage effects. The companion paper demonstrated the preparation and characterization of large unilamellar lipid vesicles. A dye release technique was employed to indicate the leakage of solutes from the vesicles during osmotic swelling. Changes in vesicle size were monitored by dynamic light scattering (DLS). In explaining the results, the model identifies three stages. The first phase involves differential increases in membrane tension with strain increasing in larger vesicles before smaller ones. In the second phase, the yield point for lysis (leakage) is reached sequentially from large sizes to small sizes. In the final phase, the lumen contents and the external medium partially equilibrate under conditions of constant membrane tension. When fit to the data, the model yields information on polydispersity-corrected values for membrane area compressibility, Young's modulus, and yield point for lysis.

Biomechanical Phenomena↗
PubMed1991

Identification and characterization of nonsedimentable lipid-protein microvesicles.

Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2+ to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesicles comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover.

Journal Article↗
PubMed1991

Determination of vesicle size distributions by freeze-fracture electron microscopy.

The most common electron microscopic technique for obtaining information on size distributions of uncollapsed membrane vesicles is based on the method of van Venetie (1980). This technique involves the sizing of only those vesicles that were freeze fractured at their equatorial planes. As a result, only a small number of images can be used to generate size distributions. Further, the technique is susceptible to systematic error. An alternate approach is to consider the complete distribution of image sizes and use this distribution to determine the average size and distribution of the vesicles. It is shown that the mean vesicle size is 4/pi times the mean image size. As well, a parameter, m, which can be determined from the image distribution, can be used to characterize the vesicle distribution. The advantage of this new approach is that images of all vesicles are used, leading to a statistically better determination of vesicle sizes.

Freeze Fracturing↗
PubMed1988

Dynamic instability of sheared microtubules observed by quasi-elastic light scattering.

The kinetics of microtubule reassembly was studied in vitro by quasi-elastic light scattering (QELS). When microtubules assembled in the absence of microtubule-associated proteins (MAPs) were sheared, they rapidly depolymerized, recovered, and reassembled. The mean length of the recovered microtubules was the same as that observed just before shearing, implying that on average one fragment per original microtubule survived the fragmentation and recovery. When microtubules that contained 25 percent brain MAP were sheared, the fragments did not depolymerize extensively and the average length of the fragments decreased by a factor of 3 relative to the unsheared sample. The results support the dynamic instability model, which predicts that cellular microtubules are latently unstable structures protected on their ends by stabilizing caps.

Animals↗
PubMed1986

Modulation of myelin basic protein-induced aggregation and fusion of liposomes by cholesterol, aliphatic aldehydes and alkanes.

The effect of cholesterol on myelin basic protein-induced aggregation of zwitterionic phospholipid vesicles was studied by turbidimetry, quasi-elastic light scattering and centrifugation techniques. Without cholesterol, the degree of vesicle aggregation caused by myelin basic protein is relatively low and is only slightly increased using cholesterol concentrations up to approx. 25-30 mol%. When the cholesterol content in the bilayer exceeds approx. 30 mol%, there is a dramatic increase in the susceptibility of the vesicles to aggregation in the presence of myelin basic protein. Palmitoyl aldehyde and eicosane, substances resembling products of lipid degradation, increase myelin basic protein promoted fusion of vesicles. The fusion is accompanied by increased leakage of entrapped carboxyfluorescein. In the presence of cholesterol, myelin basic protein-induced fusion of the liposomes becomes much more sensitive to the presence of aliphatic aldehydes or alkanes. The results suggest that cholesterol has an important role in promoting membrane adhesion in biological systems but these structures become unstable in the presence of small amounts of products of lipid degradation. The findings have important implications to the understanding of the stability of the myelin membrane.

Aldehydes↗
PubMed1985

Physical aspects of the inhibition of enzymes by hydrocarbons: the inhibition of alpha-chymotrypsin by chlorinated aromatics and alkanes.

The inhibition of alpha-chymotrypsin by a series of chlorinated hydrocarbons, including polychlorinated biphenyls, has been studied. The solubility of the hydrocarbons was determined by autocorrelation analysis of light scattering. Kinetic analysis indicated that inhibition of the enzyme occurs when 1-2 molecules of inhibitor bind per molecule of enzyme. Chlorinated aromatics including polychlorinated biphenyls were more potent than monochloroalkanes in inhibition of the enzyme. Considerable inhibition was seen when some compounds were present as micelles. Molar volume correlations suggest that chlorinated hydrocarbons exert effects on soluble enzymes similar to their effects on membrane-bound enzymes, and that a membrane lipid phase is not essential for this type of inhibition.

Chymotrypsin↗
PubMed1984

Intra-articular corticosteroid- and exercise-induced arthropathy in a horse.

Methylprednisolone acetate was injected repeatedly into both intercarpal joints of a horse that had a 3rd carpal bone fracture in 1 limb. Synovial fluid from intercarpal and radiocarpal joints of both limbs were obtained serially for study. Arthropathy developed in the fractured joint following prolonged corticosteroid therapy and exercise. In the corticosteroid-injected normal joint, the hyaluronic acid concentration initially decreased, then increased. A depletion in cartilage matrix was seen at necropsy, 175 days after onset of treatment. Determination of total protein content in synovial fluid gave the earliest indication of impending arthropathy, but viscosity studies in association with determination of hyaluronic acid concentration gave a definitive assessment of joint integrity.

Animals↗
PubMed1983

A low angle quasi-elastic light scattering investigation of Chlamydomonas reinhardtii.

This paper outlines the results of the first homodyning low angle scattering experiment known to have been reported for motile particles. Low angle scattering QELS studies were carried out on a wild strain of Chlamydomonas reinhardtii at scattering angles of 3.3 degrees, 2.6 degrees and 1.7 degrees. The resultant experimental autocorrelation functions and scaling curves were compared with a theoretical model that had been successful previously at scattering angles greater than 15 degrees.

Chlamydomonas↗
PubMed1983

A quasi-elastic light scattering and cinematographical comparison of three strains of motile Chlamydomonas reinhardtii: a wild type strain, a colchicine resistant mutant and a backward swimming mutant.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of three strains of Chlamydomonas reinhardtii: a wild cell type (WI), a colchicine resistant mutant (CO), and a backward swimming mutant (BA). Mathematical models were developed which reproduced the experimental autocorrelation functions for each of the three types of cells. These models were based on the physical characteristics of the cells' motion and the values of the parameters in the models were compared with those values that could be obtained from cinematography. Mean progressive speeds for these cells were found to be 84 microns s-1 (WI), 70 microns s-1 (CO) and 30 microns s-1 (BA).

Chlamydomonas↗