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J P Segrest

Publications and source records attributed to J P Segrest.

At least 91 records · Page 5Linked to original sources
PubMed1990

Antiviral effects of apolipoprotein A-I and its synthetic amphipathic peptide analogs.

Apolipoprotein A-I (apo A-I), the major protein component of serum high density lipoproteins, was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations. An 18 amino acid-long synthetic amphipathic alpha-helical peptide analog of apo A-I (18A) was also found to inhibit HSV-induced cell fusion at similar concentration (approximately 2 microM). Dimers of 18A connected via a proline (37pA) or an alanine (37aA) residue also inhibited virus-induced cell fusion at similar concentration, suggesting that the presence of a proline turn does not influence the antiviral activity of the amphipathic peptides. However, a peptide analog 18R, in which the distribution of charged residues was reversed, inhibited virus-induced cell fusion only at a higher (approximately 125 microM) concentration, suggesting that the anti-viral activity of the amphipathic peptide is strongly influenced by the nature of the charge distribution at the polar-nonpolar interface. Consistent with their ability to inhibit virus-induced cell fusion, the peptides inhibited the spread of HSV infection as demonstrated by a 10-fold reduction in the virus yield, when virus-infected cells were maintained in the presence of amphipathic peptides. The amphipathic peptides also inhibited penetration of virus into cells, but did not exert any effect on virus adsorption. A nearly complete inhibition of virus penetration was observed when the virus, or both virus and cells, was pretreated with the peptide, suggesting that the peptides may have a direct effect on the virus. The results indicate that amphipathic helices may be useful in designing novel antiviral agents that inhibit penetration and spread of enveloped viruses.

Amino Acid Sequence↗
PubMed1990

Charge distributions and amphipathicity of receptor-binding alpha-helices.

Contacts between ligands and cell-surface receptors result in cellular activation. Defining principles which govern these important interactions are of interest and might facilitate pharmacologic intervention. We examined receptor-binding alpha-helical segments of polypeptide hormones and globular proteins for distinguishing amino acid content and distributions. There was a slight excess of basic residues in both sets of alpha-helices compared with a panel of control helices. Helical concentrations of charged residues were quantitated using the hydrophobic moment algorithm, adapted to obtain the vector sum of side chain charges. By this analysis we detected increased concentrations of the set of basic residues (arginine, lysine and histidine) on one side of the receptor-binding alpha-helices of the polypeptide hormones, and to a lesser extent the protein ligands. Comparable data were obtained for "lytic" venom peptides and calmodulin-regulated kinase segments. There was an even greater correlation between receptor-associating alpha-helical segments and large hydrophobic moments. Receptor-binding helical segments of polypeptide hormones, and to a lesser extent those of protein ligands, often are basic and amphipathic.

Amino Acids↗
PubMed1990

Lipoprotein and oxygen transport alterations in passive smoking preadolescent children. The MCV Twin Study.

We investigated the cardiovascular effects of lifelong passive cigarette smoke exposure in preadolescent children and examined the following questions: 1) Is systemic oxygen transport altered? 2) Are coronary heart disease risk factors adversely affected? We recruited 216 families from the MCV Twin Study; 105 had at least one smoking parent. Serum thiocyanate and cotinine levels were used as measures of smoke exposure in the children and thiocyanate was proportional to the number of parental cigarettes smoked each day (p = 0.0001). Paternal smoking had no effect on these measures. Whole blood 2,3-diphosphoglycerate was higher in smoke-exposed than unexposed children (p less than 0.01) and was related to the thiocyanate level (p less than 0.02). High density lipoprotein (HDL) cholesterol was lower in passive smoking children (p less than 0.05); the HDL2 subfraction was reduced in passive smoking boys, while the HDL3 subfraction was reduced in passive smoking girls. Significant adverse alterations in systemic oxygen transport and lipoprotein profiles are already present in preadolescent children exposed to long-term passive cigarette smoke, primarily from maternal smoke. Children with long-term exposure to passive smoke may be at elevated risk for the development of premature coronary heart disease.

2,3-Diphosphoglycerate↗
PubMed1990

Apolipoprotein A-I and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation.

The envelope (membrane) glycoprotein of HIV is essential for virus attachment and entry into host cells. Additionally, when expressed on the plasma membrane of infected cells, the envelope protein is responsible for mediating cell-cell fusion which leads to the formation of multinucleated giant cells, one of the major cytopathic effects of HIV infections. The envelope glycoproteins of HIV contain regions that can fold into amphipathic alpha-helixes, and these regions have been suggested to play a role in subunit associations and in virus-induced cell fusion and cytopathic effects of HIV. We therefore tested the possibility that amphipathic helix-containing peptides and proteins may interfere with the HIV amphipathic peptides and inhibit those steps of HIV infection involving membrane fusion. Apolipoprotein A-I, the major protein component of high density lipoprotein, and its amphipathic peptide analogue were found to inhibit cell fusion, both in HIV-1-infected T cells and in recombinant vaccinia-virus-infected CD4+ HeLa cells expressing HIV envelope protein on their surfaces. The amphipathic peptides inhibited the infectivity of HIV-1. The inhibitory effects were manifest when the virus, but not cells, was pretreated with the peptides. Also, a reduction in HIV-induced cell killing was observed when virus-infected cell cultures were maintained in presence of amphipathic peptides. These results have potential implications for HIV biology and therapy.

Amino Acid Sequence↗
PubMed1989

Synthetic amphipathic peptides resembling apolipoproteins stimulate the release of human placental lactogen.

Previous studies from our laboratory demonstrated native high density lipoproteins and apolipoproteins AI, AII, and CI, stimulate the release of human placental lactogen (hPL) from trophoblast cells in culture. To examine the mechanisms by which these apolipoproteins stimulate hPL release, we have studied hPL secretion in response to several synthetic peptide analogs of the amphipathic helical structure of the apolipoproteins. The magnitude of the stimulation of hPL release in response to the analog peptides correlated with the ability to displace apolipoproteins from high density lipoprotein and with other measures of phospholipid binding affinity such as the increase in alpha-helicity and the size of complexes formed between the peptide and phospholipid. The correlation of stimulatory ability and lipid affinity suggests that the action of the apolipoproteins on hPL release may be mediated through an interaction with plasma membrane phospholipids.

Amino Acid Sequence↗
PubMed1989

Properties of lipid complexes with amphipathic helix-forming peptides. Role of distribution of peptide charges.

The peptides [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A are 18-residue peptides designed to form amphipathic helices with opposite charge distribution; [Glu1,8,Leu11,17] 18A having positively charged residues at the hydrophobic/hydrophilic interface. Both [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A strongly disrupt the bilayer structure as indicated by the relatively narrow lipid 1H and 31P NMR peaks. In addition, the 1H chemical shift of the quaternary ammonium methyl groups indicates that [Glu1,8,Leu11,17] 18A forms smaller lipoprotein particles with dimyristoylphosphatidylcholine (DMPC) than does [Glu4,9,Leu11,17] reverse-18A. However, motional properties of the lipid head group indicate that no specific salt bridges are formed between the phospholipid head group and the side chains of polar amino acids of either of the two peptides. In addition, the acyl chain conformation for the DMPC complexes with [Glu1,8,Leu11,17] 18A and with [Glu4,9,Leu11,17] reverse-18A are indistinguishable by the criterion of IR spectroscopy. The 2H linewidth of the solvent 2H2O remains narrower in frozen solutions of the DMPC-[Glu1,8,Leu11,17] 18A complexes suggesting the presence of more unfrozen bound water in this case. The two peptides exhibit many similarities in their interaction with lipids. However, [Glu1,8,Leu11,17] 18A can more readily lyse vesicles and activate lecithin:cholesterol acyltransferase. These differences do not appear to result from differences in specific charge interactions between the lipid and peptide but may be manifested through differences in hydration properties.

Calcium↗
PubMed1989

Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm.

Eisenberg's helical hydrophobic moment (less than mu H greater than) algorithm was applied to the analysis of the primary structure of amphipathic alpha-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue less than mu H greater than for the amphipathic helical and control peptides were 0.46 (+/-/-0.07) and 0.33 (0.07) (P + 0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular less than mu H greater than was plotted vs less than mu H greater than. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic alpha helix at residues 4-21. This computer-based method may enable rapid identification of peptides of the amphipathic alpha-helix class.

Algorithms↗
PubMed1989

Lipolytic surface remnants of triglyceride-rich lipoproteins are cytotoxic to macrophages but not in the presence of high density lipoprotein. A possible mechanism of atherogenesis?

Hypertriglyceridemic (HTG) serum, lipolyzed in vitro by purified bovine milk lipoprotein lipase, was found to be cytotoxic to cultured macrophages. Surviving macrophages contained numerous lipid inclusions similar to those found in foam cells. Individual lipoprotein fractions isolated from the lipolyzed HTG serum, including HDL, were also cytotoxic. Lipolysis of isolated lipoprotein fractions (either HTG or normal) allowed localization of cytotoxicity to postlipolysis remnant VLDL and chylomicron particles. The presence of a critical concentration of HDL in either the lipolysis mixture or the culture dishes inhibited the cytotoxicity. Below this critical concentration HDL itself became cytotoxic, producing lipid inclusions in surviving macrophages. The lipid fraction of the cytotoxic remnants contained the cytotoxic factor(s); neither FFA nor lysolecithin alone could account for this cytotoxicity. Postprandial lipemic sera from subjects with a brisk chylomicron response, when lipolyzed in vitro, were cytotoxic to cultured macrophages; neither fasted sera from these subjects, nor postprandial sera from normolipidemic subjects with a normal chylomicron response, were cytotoxic. Postheparin (in vivo lipolyzed) serum and its isolated lipoprotein fractions obtained 30 min after heparin injection in subjects with HTG were shown to be cytotoxic to macrophages; by 60 min most of the cytotoxicity had disappeared. The postprandial and postheparin observations support an in vivo significance for remnant-associated cytotoxicity. We hypothesize that cytotoxic remnants of lipolyzed VLDL and chylomicrons may be one of the major atherogenic lipoproteins. Further, we suggest that inhibition of the cytotoxicity of these remnants may be one important way that HDL prevents atherosclerosis.

Animals↗
PubMed1988

Effect of oxidation on the properties of apolipoproteins A-I and A-II.

Purified apolipoprotein A-I has been separated by reversed-phase high performance liquid chromatography (HPLC) into multiple peaks and these peaks have been characterized. One peak, apoA-Ib had a relatively longer retention time on HPLC but its retention time could be shortened by treatment by hydrogen peroxide. CNBr cleavage studies indicated that the differences in apoA-Ib and in its oxidation product, apoA-Ia, were due to the different oxidation states of methionine. This phenomenon was also observed in apoA-II, where methionine oxidation produced two more forms of this apolipoprotein in addition to the native form. These isomers were found to have different secondary structures and affinities for lipid. Model peptide analogs of the amphipathic helix with the same sequence but with methionine and methionine sulfoxide at the nonpolar face of the amphipathic helix were synthesized and studied. It was found that the lipid affinities of these synthetic peptide isomers were very different. They also differed in their secondary structures as studied by circular dichroism (CD). We propose that methionine oxidation introduces hydrophilic residues at the nonpolar face of the amphipathic helical domains of these apolipoproteins and, therefore, alters their secondary structure and lipid affinity.

Apolipoprotein A-I↗
PubMed1987

Studies of synthetic peptide analogs of the amphipathic helix. Effect of charge distribution, hydrophobicity, and secondary structure on lipid association and lecithin:cholesterol acyltransferase activation.

Four peptides capable of forming an amphipathic alpha-helix have been synthesized and their conformational and lipid-binding properties studied. These peptides have been designed to vary the alpha-helix-forming potential as well as the charge distribution of the model peptide. The resulting peptide analogs and their complexes with dimyristoyl phosphatidylcholine were studied by using right angle light scattering, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry techniques. The four analogs, [Glu4,9, Leu11,17] (reverse-18A, [Glu4,9, Leu5,11,17] reverse-18A, [Glu1,8, Leu11,17] 18A, and [Glu1,8, Leu5,11,17] 18A were derived from a model amphipathic peptide Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) whose lipid-associating properties strongly mimic apolipoprotein A-I or derived from Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe (reverse-18A), a peptide with little affinity for lipid and having a reversed charge distribution compared to the 18A peptide. We have shown that by substituting glutamic acid and leucine for aspartic acid and alanine, respectively, in a weak lipid-associating amphipathic helix peptide, the lipid-associating ability can be increased. Thus, peptides with both kinds of charge distribution can associate with the lipid. The ability of the peptide to disrupt phospholipid bilayers, however, is higher for 18A analogs compared to the reverse-18A analogs even after increasing the helix-forming potential and hydrophobicity. In addition to forming smaller lipoprotein particles, the modified 18A analogs were much superior to the modified reverse-18A analogs in their ability to activate the enzyme lecithin:cholesterol acyltransferase. This demonstrates that the positions of charged residues in the amphipathic helix play an important role in lecithin:cholesterol acyltransferase activation.

Circular Dichroism↗
PubMed1987

The leader peptides from bacteriorhodopsin and halorhodopsin are potential membrane-spanning amphipathic helices.

We show that the N-terminal leader peptides from the bacterial membrane proteins bacteriorhodopsin and halorhodopsin can be expected to form amphipathic alpha-helics with a highly hydrophobic nonpolar face and a narrow, negatively charged polar face. This finding is discussed in terms of a model for the integration of these proteins into the bacterial membrane.

Bacteriorhodopsins↗
PubMed1987

Effect of insulin therapy on lipoproteins in non-insulin dependent diabetes mellitus (NIDDM).

Thirty patients with NIDDM and severe hyperglycemia (fasting plasma glucose greater than 200 mg/dl) were initiated on insulin therapy. Lipoprotein concentrations were measured by the Vertical Autoprofile procedure before insulin therapy and 1, 3, 6 and 26 weeks after insulin initiation. Patients were divided into 4 phenotypes based on their pretreatment lipoprotein profile: HyperVLDL (elevated VLDL), HyperLDL (elevated LDL), HyperVLDL-LDL (elevated VLDL and LDL), and non-hyperlipidemic. There were no differences in the initial fasting plasma glucose, Hgb Alc, or fasting free insulin concentrations between the groups. Both the HyperVLDL and HyperLDL groups had significantly lower HDL-C concentrations that the non-hyperlipidemic group and the HyperVLDL-LDL group had significantly higher IDL-C than any of the other groups. Insulin therapy resulted in similar decreases in fasting plasma glucose and increases in fasting free insulin concentrations in all 4 groups. HDL-C increased in all 4 groups. The most marked improvements in HDL-C were seen in the non-hyperlipidemic (+37%) and HyperLDL (+42%) groups while the HyperVLDL group had only an 18% increase. VLDL-C fell in all groups but in the HyperVLDL group it fell dramatically to almost normal levels within the first week, whereas it took 6 weeks for the HyperVLDL-LDL group to reach its VLDL-C nadir and this was still significantly higher than normal. LDL-C improved modestly in only the HyperLDL patients after 6 weeks of insulin therapy. There were no statistically significant changes in either the IDL-C or Lp(a)-C in any of the groups during insulin therapy. The changes in HDL-C and IDL-C were negatively correlated with the fasting plasma glucose and Hgb Alc but not with the free insulin concentration. We conclude that: 1) Insulin therapy can cause dramatic improvements in HDL-C and VLDL-C while it has only a mild suppressive effect on LDL-C and no statistically significant effect on IDL-C or Lp(a)-C. The degree of improvement in the lipid profiles varied considerably between the different lipid phenotypes. 2) The hyperlipidemic phenotypes seen in these patients appear to be determined primarily by factors other than the degree of hyperglycemia and hypoinsulinemia.

Blood Glucose↗
PubMed1987

Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and immunoaffinity chromatography.

Affinity columns containing anti-apolipoprotein A-I or A-II were used to fractionate plasma into subpopulations of lipoprotein particles containing: a) apoA-I [Lp(A-I)], b) apoA-I and A-II [Lp(A-I with A-II)], and c) apoA-I but no A-II [Lp(A-I without A-II)]. Single vertical spin and electron microscopy analyses of these HDL subpopulations demonstrated that acid elution from the affinity columns caused no detectable change in size and density of the three subpopulation particles. Analysis by nondenaturing gradient gel electrophoresis of the three subpopulations found in four normal subjects identified nine HDL subspecies, designated [1] through [9] in order of increasing size; [3-7] were the major subspecies. Lp(A-I with A-II) is composed primarily of subspecies [3],[5], and [6], and may contain some subspecies [2] and [7], while Lp(A-I without A-II) represents mainly [4] and [7] and the minor subspecies [1],[2],[8], and [9]. HDL subspecies [4],[5], and [6] are found in the standard sequential flotation density cuts for both HDL3 and HDL2, which illustrates the limitations of the latter terminology. Using single vertical spin ultracentrifugation, HDL fractions were located and isolated for physical and chemical analyses, including immunoassay for apoA-I, A-II, and C-II. The distribution of the Lp(A-I without A-II) particles corresponded closely to the apoC-II distribution. An apoA-I-rich, cholesteryl ester- and phospholipid-poor subspecies was identified in the dense HDL fractions. HDL subspecies [7] was found to contain at least three separate subspecies designated [7a], [7b], and [7c]. Based on these and previously published results (Brouillette, C. G., et al. 1984. Biochemistry. 23: 359-367), we propose that the HDL subspecies adjacent in size generally differ by the association/lack of association of a hinge-like domain of amphipathic helixes in a single molecule of apoA-I.

Adult↗
PubMed1985

Effects of glyburide therapy on lipoproteins in non-insulin-dependent diabetes mellitus.

Twenty-seven patients with non-insulin-dependent diabetes mellitus and fasting plasma glucose values greater than 140 mg/dl began glyburide therapy. After entry into the study, patients were classified into three phenotypes on the basis of their initial lipoprotein profile: type IV, mixed, and normal. Glyburide lowered plasma glucose and raised plasma insulin concentrations in non-insulin-dependent diabetes mellitus. Patients with type IV hyperlipoproteinemia had higher fasting free insulin concentrations before and after therapy than patients without hyperlipoproteinemia and may have a slightly reduced hypoglycemic response to glyburide therapy. The drug caused a significant reduction in very-low-density lipoprotein cholesterol in patients with non-insulin-dependent diabetes mellitus and type IV hyperlipoproteinemia, along with a variable change in high-density lipoprotein cholesterol, independent of its effect on plasma glucose.

Cholesterol↗
PubMed1985

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine.

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.

Amino Acid Sequence↗
PubMed1985

Studies of synthetic peptide analogs of the amphipathic helix. Correlation of structure with function.

The four peptide analogs of the amphipathic helix whose interactions with dimyristoyl phosphatidylcholine were described in the preceding paper were compared with apolipoproteins (apo) A-I and A-II in ability to displace native apolipoprotein from high density lipoprotein (HDL) and in ability to activate lecithin:cholesterol acyltransferase. The rank order of the ability of the four peptide analogs to displace apo-A-I from intact HDL was 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, the same order suggested in the preceding paper for relative lipid affinities. Modified HDL from which 40% of the apo-A-I had been displaced by 18A was indistinguishable from unmodified HDL in its ability to act as a lecithin:cholesterol acyltransferase substrate. This suggests that the easily displaced apo-A-I molecules in polydisperse HDL are relatively ineffectual as lecithin:cholesterol acyltransferase activators and/or 18A replaces the lecithin:cholesterol acyltransferase activity lost. The peptide analog 18A-Pro-18A was found to be a powerful activator of lecithin:cholesterol acyltransferase when incubated with unilamellar egg phosphatidylcholine (PC) vesicles, reaching 140% of the activity of apo-A-I at a 1:1.75 peptide-to-egg PC ratio. In another experiment, it was found that discoidal egg PC complexes of 18A-Pro-18A, 18A, and des-Val10-18A, formed by cholate dialysis, had 30-45% of the activity of apo-A-I/egg PC discoidal complexes, also formed by cholate dialysis, at the same peptide/lipid weight ratio. Examination of the structures formed when the 18A-Pro-18A peptide was incubated with unilamellar egg PC vesicles indicated that the ability of 18A-Pro-18A to exceed apo-A-I in lecithin:cholesterol acyltransferase activating ability is due to the spontaneous conversion by 18A-Pro-18A of egg PC vesicles to small protein annulus-bilayer disc structures. Apo-A-I, apo-A-II, nor any of the other three peptide analogs of the amphipathic helix studied were able to convert a significant fraction of egg PC unilamellar vesicles to discoidal structures.

Amino Acid Sequence↗