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D L Puppione

Publications and source records attributed to D L Puppione.

At least 19 recordsLinked to original sources
PubMed1998

Microsequencing of bovine cerebrospinal fluid apolipoproteins: identification of bovine apolipoprotein E.

In studies of bovine plasma lipoproteins, apolipoprotein E (apoE) was not found associated with alpha-lipoproteins isolated over a broad range of densities. However, studies of cerebrospinal fluid (CSF) lipoproteins from other mammals have shown that apoE is a major apolipoprotein associated with high density lipoprotein, a fact that prompted us to determine if this were also the case in bovine CSF. CSF samples were obtained from animals with a surgically implanted catheter. Most analyzed samples were obtained from cows at various stages of the postpartum period; however, a few samples also were obtained at term or during pregnancy. Analyses of isolated ultracentrifugal fractions by polyacrylamide gel electrophoresis revealed the presence of two apo, with the expected molecular weights for apoE and apoA-I. By using both matrix-assisted laser desorption mass spectrometry and microsequencing techniques, we demonstrated that these apo are indeed apoE and apoA-I.

Amino Acid Sequence↗
PubMed1996

Chylomicron triacylglycerol fatty acids in suckling northern elephant seals (Mirounga angustirostris) resemble the composition and the distribution of fatty acids in milk fat.

Following birth, the northern elephant seal (Mirounga angustirostris) pups rapidly gain weight by ingesting milk with a high fat content, as much as 50%. To better understand the metabolism of the pups during the suckling period, the positional distributions of triacylglycerol fatty acids in both the milk and chylomicra were determined. Extracts of enzymatically digested lipids were separated by thin layer chromatography and the constituent fatty acids were separated and quantified by gas liquid chromatography. Over 84% of the fatty acids were either monoenoic or saturated, with the ratio of monoenoic to saturated fatty acids ranging between 2.9-4.0. Positional distributional analyses revealed that the very long chain monoenoics (20:1 and 22:1) were located primarily at the sn-1,3 positions of milk triacylglycerols. In the interval between the onset of lactation to the time of weaning, the content of these very long chain monoenoic fatty acids at the sn-1,3 positions increased from 13-37%. At the sn-2 position, the percentage of 18:1 was 3-5-fold higher than 16:1. Analyses indicated that the triacylglycerols in both milk and suckling pup chylomicra were similar. This particularly was true for the distributions at the sn-2 position, indicating that milk fats are being absorbed primarily via the 2-monoacylglycerol pathway.

Animals↗
PubMed1995

Identification of apolipoprotein B100 polymorphisms that affect low-density lipoprotein metabolism: description of a new approach involving monoclonal antibodies and dynamic light scattering.

Rare mutations in apolipoprotein B (apoB) can cause defective binding of low-density lipoproteins (LDLs) to the LDL receptor, leading to elevated plasma cholesterol levels and premature atherosclerosis. This communication describes a novel approach to study the effects of apoB mutations on LDL metabolism. Monoclonal antibody MB19 identifies a common polymorphism in apoB, an Ile/Thr substitution at residue 71, by binding with a 60-fold higher affinity to apoB(Ile71)-containing LDL. Because each LDL contains a single apoB, a maximum of two LDLs may be bound by the bivalent monoclonal antibody. Thus, at the appropriate concentration, an equivalent amount of MB19 will promote substantial dimer formation of LDL containing the strongly binding apoB(Ile71), but little dimer formation of LDL containing the weakly binding apoB(Thr71). For LDL isolated from heterozygous individuals, the amount of dimer formed, determined by dynamic light scattering, yields an estimate of the allelic ratio of the two forms of LDL. For such individuals, not only the effect of the polymorphism recognized by MB19 but also the effects of other polymorphisms on the LDL allelic ratio can be determined. Examination of six normolipemic MB19 heterozygotes gave percent allelic ratios between 48:52 and 51:49 tight:weak-binding LDL, not significantly different from a 50:50 ratio. These individuals were also heterozygous for six common apoB polymorphisms, allowing calculation of the odds that each of these polymorphisms caused significant alterations in lipid levels. In contrast, the rare mutation at residue 3500 causing defective binding to the LDL receptor and familial defective apoB100 (FDB) resulted in substantial changes (26:74 and 13:87) in LDL allelic ratio in both of two FDB individuals examined.

Alleles↗
PubMed1994

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci.

We have tested for evidence of linkage between the genetic loci determining concentrations and composition of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes participating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple sequence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determining plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) locus on chromosome 6 (P = 0.02). Thus, these data indicate the simultaneous involvement of at least two different genetic loci in the determination of the levels of HDL and its associated lipoproteins.

Apolipoprotein A-II↗
PubMed1994

A microprecipitation technique suitable for measuring alpha-lipoprotein cholesterol.

A semi-automated method has been developed for determining alpha-lipoprotein cholesterol values. Precipitation of apolipoprotein B containing lipoproteins takes place in wells of microtiter plates after 100 microL of serum are mixed with 20 microL of a heparin/MnCl2 solution. A Beckman (Fullerton, CA) Biomek 1000 work station is used to transfer sera, supernatants and reagents between tubes and microtiter plates. Supernatant cholesterol is determined enzymatically, and absorbances are read at 490 nm using a Molecular Devices Corporation (Palo Alto, CA) plate reader. Values obtained on both fresh and frozen serum samples agreed with corresponding data obtained at the Centers for Disease Control (CDC; Atlanta, GA). For the fresh samples, the average bias was 2.87%. The within-run coefficients of variations were between 2.2 and 0.6% for the data obtained on CDC frozen control pools. The results indicate that the semi-automated method is suitable for obtaining accurate and precise data for alpha-lipoprotein cholesterol. The method lends itself to the analysis of large numbers of samples and is particularly suited for the study of lipoproteins of small mammals.

Autoanalysis↗
PubMed1993

Evidence for linkage of the apolipoprotein A-II locus to plasma apolipoprotein A-II and free fatty acid levels in mice and humans.

Although it has been hypothesized that the synteny between mouse and human genes provides an approach to the localization of genes that determine quantitative traits in humans, this has yet to be demonstrated. We tested this approach with two quantitative traits, plasma apolipoprotein A-II (apoAII) and free fatty acid (FFA) levels. ApoAII is the second most abundant protein of high density lipoprotein particles, but its function remains largely unknown. We now show that, in a backcross between strains Mus spretus and C57BL/6J, apoAII levels correlate with plasma FFA concentrations on both chow (P < 0.0001) and high-fat (P < 0.0003) diets and that apoAII levels are linked to the apoAII gene (P < 0.0002). To test whether variations of the apoAII gene influence plasma lipid metabolism in humans, we studied 306 individuals in 25 families enriched for coronary artery disease. The segregation of the apoAII gene was followed by using an informative simple sequence repeat in the second intron of the gene and two nearby genetic markers. Robust sib-pair linkage analysis was performed on members of these families using the SAGE linkage programs. The results suggest linkage between the human apoAII gene and a gene controlling plasma apoAII levels (P = 0.03). Plasma apoAII levels were also significantly correlated with plasma FFA levels (P = 0.007). Moreover, the apoAII gene exhibited linkage with a gene controlling FFA levels (P = 0.003). Evidence for nonrandom segregation was seen with markers as far as 6-12 centimorgans from the apoAII structural locus. These data provide evidence, in two species, that the apoAII gene is linked to a gene that controls plasma apoAII levels and that apoAII influences, by an unknown mechanism, plasma FFA levels. The results illustrate the utility of animal studies for analysis of complex traits.

Animals↗
PubMed1993

Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice.

The role of apolipoprotein A-II (apoA-II) in the metabolism of high density lipoproteins (HDL) is poorly understood. Previous studies of naturally occurring variations of apoA-II in mice have, however, suggested that apoA-II expression influences HDL size and concentration. We now provide definitive evidence of this using transgenic mice overexpressing mouse apoA-II. Thus, as compared with nontransgenic littermates, transgenic mice possessing 4-fold elevations of apoA-II mRNA and 2-3-fold elevations in plasma apoA-II levels exhibited more than a 2-fold increase in HDL levels as well as about a 25% increase in average HDL diameter. Overexpression of mouse apoA-II also resulted in a substantial decrease in the levels of apolipoprotein E associated with HDL, suggesting that apoA-II can displace apoE from HDL. The apoA-II transgenic mice also exhibited a 2-4-fold increase in plasma very low density and low density lipoprotein-cholesterol levels, suggesting novel interactions among the classes of plasma lipoproteins.

Animals↗
PubMed1992

Positional analyses of triacylglycerol fatty acids in the milk fat of the antarctic fur seal (Arctocephalus gazella).

The positional distribution of fatty acids has been determined for the milk triacylglycerols of the Antarctic fur seal, Arctocephalus gazella. Of particular interest was the positional distribution of the polyunsaturated n-3 fatty acids in milk triacylglycerols (TG). In adipocytes of pinnipeds, TG are synthesized with the n-3 fatty acids primarily in the sn-1,3 positions. To determine the positional distribution, extracts of enzymatically digested lipids were separated by thin-layer chromatography, and the constituent fatty acids were separated and quantified by gas-liquid chromatography. Monoenoic and saturated fatty acids comprised over 75% of the total, the ratio of monoenoic to saturated fatty acids being 2:1. The percent content of the long-chain n-3 fatty acids, 20:5, 22:5 and 22:6, ranged between 15-20%. The positional analyses revealed that at the sn-2 position of milk TG, saturated fatty acids were in excess (57%), and the content of n-3 fatty acids was less than 5%. More than 80% of the n-3 fatty acids in milk were located in the sn-1,3 positions. The data indicate that in pinnipeds TG are synthesized in the mammary gland and adipose tissue with fatty acids having similar positional distributions.

Adipose Tissue↗
PubMed1992

Identification of a neutral lipid core in a transiently expressed and secreted lipoprotein containing an apoB-48-like apolipoprotein.

The presence of core lipids in lipoproteins expressed and secreted by transfected HepG2 cells was demonstrated by measuring the densities of these lipoproteins before and after treatment with a bacterial lipase specific for neutral lipids. HepG2 cells were reproducibly transfected with pRSV/B48, containing a truncated human apolipoprotein B-100 (apoB-100) cDNA (nucleotides 1 to 6860, where nucleotide 129 is the start of translation). Northern blots of cellular message probed with apoB-48 showed abundant transcription of an apoB-48-sized message as well as endogenous apoB-100 message. When grown in the presence of [35S]methionine, pRSV/B48-transfected cells secreted lipoproteins containing an apoB-48-like apolipoprotein. This lipoprotein banded at a density of 1.11 g/ml in isopycnic NaBr gradients. Electron microscopy of the apoB-48-containing lipoproteins demonstrated spherical particles with an average diameter of 124A. A sedimentation rate of 8.4S was measured by sucrose gradient sedimentation. When the apoB-48-containing particles were treated with a bacterial lipase (from Chromobacterium viscosum), shown to hydrolyze triglycerides and cholesteryl esters but not phospholipids, their density increased to 1.18 g/ml, consistent with removal of core lipids. When the secreted lipoprotein was modeled as a spherical particle containing a single molecule of apoB-48, a triglyceride-filled core, and a surface monolayer of phospholipid and protein, the hydrodynamic properties were consistent with the observed sedimentation coefficient, buoyant densities before and after lipase treatment, and the diameter as seen with the electron microscope. These data indicate that transfected HepG2 cells assembled and secreted lipoproteins possessing the same physical structure as naturally occurring lipoproteins.

Apolipoprotein B-48↗
PubMed1989

Low density lipoprotein heterogeneity in the cebus monkey.

In studies of cebus monkey plasma lipoproteins, we have used an ultracentrifugally generated density gradient to isolate two distinct species of low density lipoproteins (LDL). Compositional analyses revealed that each of the ultracentrifugally isolated fractions was enriched in cholesteryl esters and contained a single apolipoprotein which in terms of its mobility on SDS gels corresponded to apolipoprotein B-100, the major apolipoprotein of human LDL. Hydrodynamic measurements carried out in the analytical ultracentrifuge showed that F1.20 values were 30.0 for LDL1 and 23.5 for LDL2. In a solution of density 1.0069 g/ml, the sedimentation rates were 5.9 and 7.2 S for LDL1 and LDL2, respectively. In addition to sedimentation velocity data, we describe a new approach for using these same data to obtain calculated values for molecular weight. The hydrated densities calculated for the two fractions were 1.033 and 1.045 g/ml and calculated molecular weights were 3.08 million for LDL1 and 2.42 million for LDL2. Hydrated density values were in excellent agreement with those calculated from compositional data. Electron microscopy data showed that LDL1 had a larger mean diameter of 26.7 nm than LDL2 which had a diameter of 19.3 nm. Native gel electrophoretic analyses of the two LDL fractions in 3.5% acrylamide showed that, consistent with its size, LDL1 had slower mobility than LDL2.

Animals↗
PubMed1988

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B.

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.

Amino Acids↗
PubMed1986

Physiochemical study of rock crab lipoproteins.

Physicochemical studies have been carried out on the hemolymph and egg lipoproteins of the rock crab (Cancer antennarius). Analytical ultracentrifugal analyses of vitellogenic female HDL3 revealed the presence of two types of lipoproteins. The first with a sedimentation rate of 5.35 S was comparable to lipoproteins in male and non-vitellogenic female hemolymph. The second with a sedimentation rate of 10.74 S was comparable to the major lipoprotein of egg yolk. A similar comparison could be made following electrophoretic analyses in native polyacrylamide gels. Electrophoresis in SDS-polyacrylamide gels revealed three major apolipoproteins common to egg and vitellogenic HDL3. A fourth apolipoprotein was found in both male and female HDL3. In contrast to mammalian HDL, none of these crustacean apolipoproteins had a molecular weight less than 82 000. One of these apolipoproteins appears to be comparable physicochemically to the enteric form of apolipoprotein B in mammals.

Animals↗
PubMed1985

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII).

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.

Animals↗
PubMed1983

Genetic control of lipid transport in mice. I. Structural properties and polymorphisms of plasma lipoproteins.

To examine genetic factors involved in mammalian lipid transport, we present a detailed analysis of the plasma lipoproteins of the mouse and a survey of these lipoproteins among different inbred strains for genetic variations. Plasma lipoproteins were separated by differential ultracentrifugation into three density fractions: with densities less than 1.006 g/ml, between 1.006 and 1.063 g/ml, and between 1.063 and 1.21 g/ml. On agarose gels, the d less than 1.006 g/ml fraction migrated as a single major band with pre-beta mobility and appeared to correspond to human very low density lipoproteins based on its lipid and protein composition. The intermediate density class was heterogeneous, containing particles with beta and alpha mobilities on agarose gels. The 1.063-1.21 g/ml density fraction exhibited a single alpha-migrating band on agarose gels, characteristic of high density lipoprotein in other mammalian plasma. Marked genetic variation was seen for lipoprotein mobilities among different inbred strains. In Swiss random bred mice, most of the apoprotein was in high density lipoprotein (104 mg/dl of plasma). Lower protein concentrations of 8.5 and 6.2 mg/dl were found in the d = 1.006-1.063 g/ml class and in the very low density lipoprotein, respectively. Proteins thought to be homologous to human apolipoproteins A-I, A-II, B, and E were identified on the basis of their distributions among the lipoprotein fractions, their molecular weights and isoelectric points and, for A-I, B, and E, by their cross-reactivities with antibodies prepared against purified rat apolipoproteins. Considerable structural and quantitative variations also exist among these apolipoproteins isolated from different inbred mouse strains. Our results provide a basis for the use of the mouse as a model system for the study of the genetic control of lipid transport.

Animals↗
PubMed1982

Characterization of unusual intermediate density lipoproteins.

We report on the physicochemical properties of unusual lipoproteins isolated from both lymph and blood of ruminating cattle. The densities of most of these particles fall within the range between 1.006 and 1.020 g/ml, although densities of 0.97-0.99 g/ml are calculated from chemical composition, assuming a liquid core. The triglycerides of these particles have a high content of saturated fatty acids. The major apoprotein has a mobility on polyacrylamide-SDS gels consistent with a molecular weight of 40,000. The negatively-stained particles appear flattened and asymmetric in electron micrographs. The particles are very large, with molecular weights in the 20 to 250 million dalton range, and they scatter light strongly. The hydrodynamic frictional ratio is about 1.4, consistent with oblate ellipsoids with axial ratios of about 8 to 1. The flat appearance, asymmetric shape, and anomalous densities of the particles would be explained if these lipoproteins consisted of a core of crystallized triglycerides encapsulated within a phospholipid monolayer. Crystallization of the saturated triglycerides could occur during routine lipoprotein isolation, in which temperatures much lower than the melting points of their core lipids are employed. when protocols are done entirely at 37 degrees C, the unusual structures are not observed in the intermediate density class. Although the saturated fats in these bovine lipoproteins are derived from ruminal fermentation, we feel that any triglyceride-rich lipoprotein highly enriched in saturated fats will behave similarly if isolation temperatures are well below the melting points of the core lipids.

Animals↗
PubMed1982

Physicochemical characterization of ten fractions of bovine alpha lipoproteins.

With the onset of milk production, serum concentrations of alpha lipoproteins in the dairy cow steadily increase, frequently attaining values greater than 1.5 g/dl. Since these lipoproteins comprise a highly polydisperse system, we have carried out studies to explore differences among bovine alpha lipoproteins in the density interval between 1.05 to 1.21 g/ml. Separation into ten fractions was achieved ultracentrifugally in an isopycnic gradient. Agarose gel electrophoresis showed that all but the bottom fraction contained alpha lipoproteins as either the major or sole lipoprotein class. Compositional analyses revealed an increasing percentage of both protein and phospholipid and a decreasing percentage of cholesterol with increasing fraction density. The esterified to unesterified cholesterol ratio ranged from 3 to 8 from the top to the bottom of the gradient. The densities of the particles obtained from the various fractions were calculated both from sedimentation velocity measurements and from compositional analyses. The resulting density values agreed well with the solution densities of these isopycnic gradient fractions. The major apoprotein of each fraction was apoA-I. Combining diffusion coefficient data obtained by intensity fluctuation spectroscopy with sedimentation velocity data, we were able to calculate molecular weights, frictional ratios, and Einstein-Stokes radii for three of the fractions. Results are discussed in terms of previously published data on bovine lipoproteins as well as other mammalian data.

Animals↗