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Biomedical subjects

J P Segrest

Publications and source records attributed to J P Segrest.

At least 109 records · Page 6Linked to original sources
PubMed1984

Structural studies of apolipoprotein A-I/phosphatidylcholine recombinants by high-field proton NMR, nondenaturing gradient gel electrophoresis, and electron microscopy.

Complexes formed between apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) or egg phosphatidylcholine have been studied by high-field 1H NMR, nondenaturing gradient gel electrophoresis, electron microscopy, and gel filtration chromatography. Emphasis has been placed on an analysis of the particle size distribution within the micellar complexes produced at lipid/protein molar ratios of 40-700. As determined by electron microscopy and gel filtration of DMPC/apo A-I complexes, the size of the discoidal micelles produced appears to increase uniformly with an increasing lipid/protein ratio. By electron microscopy, the diameters of isolated DMPC/apo A-I discoidal micelles range from approximately 89 A at a 40 molar ratio to 205 A at a 700 molar ratio. Analysis of the micellar complexes by 1H NMR shows that concomitant with the increase in size is the progressive downfield shift of the choline N-methyl proton resonance of the complex which is observed from 3.245 to 3.267 ppm over the above molar ratio range. The relationship between chemical shift and micelle size is most simply interpreted as arising from a weighted averaging of two lipid environments--lipid-lipid and lipid-protein. In contrast to the above interpretation of the gel filtration experiments on DMPC/apo A-I complexes, nondenaturing gradient gel electrophoresis analysis of particle size distribution leads to an unexpected observation: as the DMPC/apo A-I ratio increases, discrete complexes of increasing size are formed in an apparently quantized manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗
PubMed1983

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells.

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.

Animals↗
PubMed1983

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction.

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.

Cholesterol↗
PubMed1982

Structural domains of vesicular stomatitis virus. A study by differential scanning calorimetry, thermal gel analysis, and thermal electron microscopy.

Differential scanning calorimetry has been used in combination with thermal gel analysis and electron microscopy to identify and study the structural domains of the membrane-enclosed vesicular stomatitis virus and its isolated internal components. Three major endothermic transitions centered at approximately 52, 76, and 80 degrees C and at least two minor transitions are observed at pH 7.0 for the intact virion. Thermal gel analysis suggests the possibility that specific proteins of vesicular stomatitis virus are involved in two or more of the calorimetric transitions. The effect of heating to defined temperatures on the morphology of the virion was studied by negative stain electron microscopy. The results of these "thermal EM" studies show discrete irreversible morphological changes in the virion which seem to coincide with the three major calorimetric transitions.

Animals↗
PubMed1982

Computerized rapid high resolution quantitative analysis of plasma lipoproteins based upon single vertical spin centrifugation.

A method has been developed for rapidly quantitating the cholesterol concentration of normal and certain variant lipoproteins in a large number of patients (over 240 in one week). The method employs a microcomputer interfaced to the vertical autoprofiler (VAP) described earlier (Chung et al. 1981. J. Lipid Res. 22: 1003-1014). Software developed to accomplish rapid on-line analysis of the VAP signal uses peak shapes and positions derived from prior VAP analysis of isolated authentic lipoproteins HDL, LDL, and VLDL to quantitate these species in a VAP profile. Variant lipoproteins VHDL (a species with density greater than that of HDL(3)), MDL (a species, most likely Lp(a), with density intermediate between that of HDL and LDL), and IDL are subsequently quantitated by a method combining difference calculations with curve shapes. The procedure has been validated qualitatively by negative stain electron microscopy, gradient gel electrophoresis, strip electrophoresis, chemical analysis of the lipids, radioimmunoassay of the apolipoproteins, and measurement of the density of the peak centers. It has been validated quantitatively by comparison with Lipid Research Clinic methodology for HDL-, LDL-, and VLDL-cholesterol, and for MDL- and IDL-cholesterol by comparison of the amounts of MDL or IDL predicted to be present by the method with that known to be present following standard addition to whole plasma. These validations show that the method is a rapid and accurate technique of lipoprotein analysis suitable for the routine screening of patients for abnormal amounts of normal or variant lipoproteins, as well as for use as a research tool for quantitation of changes in cholesterol content of six or seven different plasma lipoprotein fractions.-Cone, J. T., J. P. Segrest, B. H. Chung, J. B. Ragland, S. M. Sabesin, and A. Glasscock. Computerized rapid high resolution quantitative analysis of plasma lipoproteins based upon single vertical spin centrifugation.

Apolipoproteins↗
PubMed1981

High resolution plasma lipoprotein cholesterol profiles by a rapid, high volume semi-automated method.

A new rapid and sensitive method, the single vertical spin autoprofiler (VAP), has been developed for quantitative profiling of the major plasma lipoproteins. The method involves a combination of single vertical spin separation of plasma and continuous on-line analysis of cholesterol. Plasma lipoproteins are first separated by a 45-min spin in a vertical rotor, after which the amount of cholesterol in the effluent of each tube is monitored continuously by a modification of the BMC automated enzymatic cholesterol method; simultaneously, 80% of the sample is diverted by stream-splitting to a fraction collector for further analysis, if desired. VAP not only resolves very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) peaks quantitatively but also detects the presence of intermediate density lipoprotein (IDL) and other lipoprotein variants. VAP was highly reproducible; the inter-run coefficient of variation for cholesterol concentration in VLDL, LDL, and HDL was 4.8%, 2.9%, and 2.4%, respectively. Cholesterol recovery using VAP was 98.5 +/- 3.5%. Lipoprotein-cholesterol profiles of plasma from three major hyperlipoproteinemia phenotypes examined by VAP were qualitatively and quantitatively different from each other and from profiles of normolipidemic individuals. One significant finding was that IDL could be detected in the plasma of all type IV hyperlipoproteinemic subjects examined thus far. Several variant lipoprotein profiles which did not correspond to known phenotypes have also been detected by VAP using plasma from hyperlipidemic as well as from normolipidemic subjects. We submit that VAP is an accurate and rapid method for lipoprotein analysis, either for routine clinical screening or for detailed experimental studies. In addition, VAP provides a visual display of partially to completely resolved lipoprotein classes that is suitable for computer-assisted analysis.-Chung, B. H., J. P. Segrest, J. T. Cone, J. Pfau, J. C. Geer, and L. A. Duncan. High resolution plasma lipoprotein cholesterol profiles by a rapid, high volume semi-automated method.

Animals↗
PubMed1980

Studies of synthetic peptide analogs of the amphipathic helix. Effect of charged amino acid residue topography on lipid affinity.

The amphipathic helix hypothesis for plasma lipoproteins was investigated using synthetic peptides. The lipid-associating properties of two potentially amphipathic model peptides and two analogs were studied by incubating synthetic peptides with small unilamellar vesicles and protein-lipid association examined by equilibrium density centrifugation, leakage of liposome-entrapped fluorescence compounds, intrinsic tryptophan fluorescence, and circular dichroism spectroscopy. The analog peptides were designed to determine the significance of the number and specific location of the charged residues in amphipathic domains of plasma lipoproteins to protein-lipid association. Based on the four procedures used to examine protein-lipid interactions, the two model peptides (18Aa, 18As) were found to associate strongly with liposomes; the two analog peptides (18As1, 18Asr), differing only with respect to the number and/or position of their charged residues, failed to demonstrate similar lipid binding properties. These findings support the earlier suggestions of the importance of the charged residues, but do not define the precise mechanisms involved. Such amino acids may help initiate the lipid-protein association by electrostatic interactions, contribute to the hydrophobicity of the nonpolar face of the helix by the acyl portion of lysine and arginine, and/or complement the charge distribution in the polar head regions of the phospholipid molecules.

Amino Acid Sequence↗
PubMed1980

Detergent removal during membrane reconstitution.

Efficiency of detergent removal during the course of several different procedures for membrane protein reconstitution was examined. Reconstitution methods studied include ethanol injection-dialysis, detergent dialysis and detergent-gel filtration. In the ethanol injection-dialysis method, approx. 70 molecules of ethanol per 1000 molecules of phospholipid are retained even after extensive (150 h) dialysis. Efficiency of detergent removal by dialysis depends on the detergent. However, even for sodium deoxycholate, a detergent possessing a large critical micelle concentration, there are approx. 7 molecules of deoxycholate per 1000 molecules of phospholipid retained by the bilayer even after extensive (310 h) dialysis. Detergent removal by gel filtration (Sephadex G-200 or G-50) of deoxycholate, cholic acid and Triton X-100 is more efficient than removal by dialysis; as few as 10 molecules of deoxycholate are retained per 100 molecules of phospholipid after one column passage, taking only a few hours. Ethanol was less efficiently removed by one passage over a Sephadex column than by extensive dialysis. Removal of Triton X-100 by passage over, or dialysis against, Biobeads SM-2 resulted in a similar level of detergent retention to that found by passage over Sephadex G-200 or G-50. Utilizing gel-filtration techniques, we have examined the competition for the hydrophobic peptide of glycophorin, T(is), between sodium deoxycholate and a series of phospholipids as a possible means of obtaining a quantitative measure of protein-lipid affinity. On the basis of these preliminary studies we conclude that the T(is) peptide has a relative lipid affinity of phosphatidylinositol > phosphatidylcholine > phosphatidylserine.

Chromatography, Gel↗
PubMed1980

Liposome-cell interactions. A rapid assay for cells in suspension culture.

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interation. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.

Animals↗
PubMed1980

Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor.

A rapid method has been developed for separation of the major plasma lipoproteins from up to 96 ml of plasma by a single ultracentrifugation step. This separation was achieved by a discontinuous density gradient centrifugation between the density range of 1.006 and 1.30 g/ml in Sorvall vertical rotors. Each lipoprotein fraction was sharply banded with VLDL at the top, LDL in the upper middle, and HDL in the lower middle portion of the tube. Use of authentic 125I-labeled lipoproteins showed that complete separation of the three major classes and partial separation of HDL2 and HDL3 was achieved. The lipoprotein fractions prepared by this technique have properties indistinguishable from those isolated by the sequential flotation method in regard to their equilibrium banding density, electrophoretic mobility, and apolipoprotein composition. This method is suitable for the preparative isolation of lipoproteins as well as for quantitative clinical determinations of cholesterol and triglycerides in VLDL, LDL and HDL fractions of plasma. Used as an analytical tool this method allows samples as small as 1 ml of plasma and spin times as short as 45 min. Cholesterol levels in HDL fractions separated by this method have significantly lower values (P less than 0.05) than those estimated by the heparin-manganese chloride precipitation method.

Centrifugation, Density Gradient↗