Biomedical subjects

J T Sparrow

Publications and source records attributed to J T Sparrow.

At least 55 records · Page 3Linked to original sources

PubMed1986

Lipid-peptide association and activation of lecithin:cholesterol acyltransferase. Effect of alpha-helicity.

A series of apolipopeptides, in which single proline substitutions were made at various sites in the 20-residue sequence, have been synthesized and tested. These peptides have nearly the same hydrophobic content, but very different helical contents, in a structure-making solvent. The affinity of these peptides for phospholipids was evaluated on the basis of their intrinsic tryptophan fluorescence and equilibrium dialysis against model high density lipoproteins. Proline substitutions at one end of the peptide had little or no effect on the fluorescence, circular dichroism, affinity for model high density lipoproteins, or activation of human plasma lecithin:cholesterol acyltransferase. By contrast, there was a dramatic change in all of these variables as the site of substitution was moved progressively closer to the middle of the peptide. All of these data suggested that a helix breaker that is substituted at the midpoint of a helical surface-associating peptide will greatly reduce its affinity for phospholipid surfaces. These results demonstrate that helicity and hydrophobicity are independent determinants of the affinity of an apolipopeptide for a phospholipid surface.

Amino Acid Sequence↗

PubMed1986

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3.

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.

Animals↗

PubMed1986

Binding of a high reactive heparin to human apolipoprotein E: identification of two heparin-binding domains.

Ligand-blotting and dot-blotting procedures were used to investigate the binding of [125I]-heparin to apolipoprotein E, its thrombin fragments E22 (residues 1-191) and E12 (residues 192-299), and to nine apolipoprotein E synthetic fragments. E22 and E12 bound [125I] heparin indicating multiple heparin-binding domains. Synthetic peptides of apoE corresponding to residues 129-169, 139-169, and 144-169, but not 148-169, bound [125I] heparin suggesting that residues 144-147 (Leu-Arg-Lys-Arg) in E22 are important for binding. Peptide 202-243 and 211-243 but not 219-243 bound [125I] heparin suggesting that residues 211-218 (Gly-Glu-Arg-Leu-Arg-Ala-Arg-Met) comprise a portion of the E12 heparin-binding domain.

Amino Acid Sequence↗

PubMed1986

Induction of anti-HIV neutralizing antibodies by synthetic peptides.

Two synthetic peptides containing amino acid sequences analogous to the envelope glycoprotein of human T-lymphotropic virus (HTLV) type III (HTLV-III) and lymphadenopathy associated virus (LAV) were produced and used to immunize rabbits. The subsequent rabbit antisera neutralized HTLV-III infectivity in vitro. The two synthetic peptides corresponded to regions associated with the gp120 or gp41 subunits respectively, of human immunodeficiency virus (HIV). This data indicates that at least two neutralizing epitopes are present on the envelope glycoprotein of HIV and these epitopes are associated with two distinct virus envelope glycoproteins. Antisera generated against these peptides neutralized infectivity of two different isolates of HTLV-III. The data is discussed in terms of possible strategy for developing an effective vaccine against the etiologic agents of acquired immune deficiency syndrome (AIDS).

Amino Acid Sequence↗

PubMed1986

Treatment of persistent pubertal gynecomastia with dihydrotestosterone heptanoate.

Four boys with persistent pubertal gynecomastia were given intramuscular dihydrotestosterone heptanoate (DHT-hp) at 2 to 4-week intervals for 16 weeks. By the end of treatment, breast size in all four boys had decreased 67% to 78%. Initial plasma levels of gonadotropins, estradiol, testosterone, and dihydrotestosterone (DHT) were normal. Mean plasma DHT concentration rose with the injections of DHT-hp, and remained elevated throughout the treatment period. Estradiol, LH, FSH, and testosterone decreased during treatment, as did 24-hour urinary LH and FSH. No regrowth of breast tissue was observed 6 to 15 months after treatment, although hormone concentrations had returned to near pretreatment values by 2 months after the last injection. DHT-hp has potential to be an effective medical therapy for persistent pubertal gynecomastia.

Adolescent↗

PubMed1986

Expression of the v-mos gene alters a Mr 55,000 protein during acute infection by Moloney murine sarcoma virus.

Infection of the rat myoblast cell line, L6E9, with Moloney murine sarcoma virus (Mo-MuSV) clone 124, altered a cellular protein of Mr 55,000 (P55) within 2 days of infection. The alteration of P55 was observed as a reduction in its steady-state level in cell extracts. The reduction of P55 correlated with the appearance of p37mos in infected cells. Except for P55 and one other protein, no change was detected in the total protein pattern of infected cells compared to uninfected cells, as judged by either immunoblots of one-dimensional NaDodSO4 gels or direct two-dimensional gel analysis. P55 levels were unchanged when L6E9 cells were infected with Moloney murine leukemia virus or several different transforming retroviruses. To determine the specificity of this v-mos-induced effect on P55, L6E9 cells were acutely infected with a temperature-sensitive variant (ts110) of Mo-MuSV. When these cells were shifted from 39 degrees C to 33 degrees C, which activates the gag-mos gene product, the P55 level dropped by greater than 50% within 2-3 hr. Conversely, with a shift in temperature from 33 degrees C to 39 degrees C, the cells' P55 level returned to normal within 5 hr, starting at 30 min after shift. These results clearly show that v-mos expression in acutely infected L6E9 cells alters the cellular protein, P55.

Animals↗

PubMed1986

In vivo interaction of synthetic acylated apopeptides with high density lipoproteins in rat.

The metabolism of synthetic peptide analogues of high density lipoprotein (HDL) apoproteins has been studied in the rat. These compounds are 15-amino acid lipid associating peptides (LAPs) bearing acyl chains of various lengths (0-16 carbon units). After injection of each 125I-LAP, the serum decay curves suggested a two-compartment process with a clearance rate decreasing when the acyl chain lengths increased. The similarity between the apparent half-life of C16-LAP and that of apoprotein A-I as well as the chromatographic analysis of rat serum were consistent with a partitioning of the LAPs between HDL and the aqueous phase. This was strongly dependent upon the acyl chain length of the LAPs. The distribution volumes of the 125I-LAPs in organs were measured 10 min after injection. The results were analyzed using a model explicitly predicting the organ distribution volumes of HDL and the equilibrium constant (Keq) of the binding of each LAP to HDL. HDL distributed significantly in the adrenals (250 microliters/g), liver (80 microliters/g), and ovaries (55 microliters/g), but not in the kidneys. This suggests that the binding of HDL apoproteins to kidneys, reported by others, was due to the uptake of free apoproteins. The Keqs exhibited a log-linear relationship with respect to the acyl chain length of the LAPs. Each carbon unit added to the acyl chain decreased the free energy of association by a constant value (0.3 kcal mol-1). This clearly showed a strict hydrophobic effect similar to that previously observed in vitro.

Animals↗

PubMed1985

Apolipoprotein E: phospholipid binding studies with synthetic peptides containing the putative receptor binding region.

To define the lipid and receptor binding regions of apolipoprotein E (apoE), we have synthesized four peptides beginning at residue 169 and continuing through the putative receptor binding region and ending at residue 129 so as to include a proposed lipid binding domain. The peptides were synthesized by solid-phase techniques, cleaved with anhydrous HF, and purified by ion-exchange and semipreparative reversed-phase high-performance liquid chromatography (HPLC). The peptides had the correct amino acid composition and were greater than 99% pure by analytical reversed-phase HPLC. The circular dichroic spectrum of each peptide was recorded before and after mixing with dimyristoylphosphatidylcholine. With apoE (148-169), apoE (144-169), and apoE (139-169), no changes were observed in the ellipticity at 222 nm. However, with apoE (129-169), an increase in alpha-helicity to approximately 42% was observed. Density gradient ultracentrifugation of the lipid-peptide mixture permitted isolation of a complex with apoE (129-169) with a molar ratio of lipid to peptide of 125:1, which was stable to recentrifugation. The alpha-helicity of the peptide in the complex was estimated to be 56%. No complexes were isolated from the gradients of the shorter peptides. Therefore, we conclude that the amphipathic helix formed by residues 130-150 contains one of the lipid binding regions of apoE.

Amino Acids↗

PubMed1985

Monoclonal antibodies against bovine milk lipoprotein lipase. Characterization of an antibody specific for the apolipoprotein C-II binding site.

Ten murine monoclonal antibodies have been produced that are specific for bovine milk lipoprotein lipase. One monoclonal antibody, bLPL-mAb-7, inhibited completely the apolipoprotein C-II (apo-C-II)-dependent enzymic hydrolysis of trioleoylglycerol in a phospholipid-stabilized emulsion, but had no effect on the hydrolysis of the water-soluble substrate p-nitro-phenylacetate. Four times more bLPL-mAb-7 was required to achieve 50% inactivation of lipoprotein lipase activity when the enzyme was preincubated with excess apo-C-II. Disruption of the binding of a dansyl-labeled apo-C-II peptide to lipoprotein lipase by bLPL-mAb-7 was demonstrated by resonance energy transfer, both in the presence and absence of lipid. This antibody thus appears to recognize the apo-C-II binding site of lipoprotein lipase. In addition, bLPL-mAb-7 also inhibited the lipoprotein lipase activity of human post-heparin plasma.

Animals↗

PubMed1985

Synthetic hepatitis B surface antigen peptide vaccine.

A synthetic hepatitis B surface antigen (HBsAg) peptide was prepared containing amino acid residues 122-137 of the major HBsAg polypeptide. This peptide was cyclized by the introduction of an intrachain disulfide bond between cysteine residues at positions 124 and 137 because previous studies had shown that intact disulfide bonds are critical for maintenance of HBsAg activity. An anti-HBs response was produced in mice by free peptide entrapped in liposomes. However, the immunogenicity was enhanced by aggregation into micelles, and by coupling to tetanus toxoid. Analysis of the peptide with a panel of monoclonal antibodies showed that peptide 122-137 contained a conformation (discontinuous) group a epitope and a sequential (continuous) subgroup y epitope. In addition, the cyclic peptide inhibited a human anti-HBs idiotype-anti-idiotype reaction with specificity for group a determinant(s). The potential for synthetic peptides for hepatitis B virus vaccine development is discussed.

Animals↗

PubMed1985

In vivo detection of human hepatoma secreting hepatitis B surface antigen in nude mice with radiolabeled monoclonal antibodies that recognize distinct epitopes.

Hepatitis B surface antigen (HBsAg), produced by a human hepatoma which had been transplanted into athymic nude mice, was specifically detected in vivo by 131I-labeled monoclonal antibodies (McAb) directed against distinct epitopes of HBsAg (anti-HBs). Significantly higher levels of radioactivity were present in the hepatoma secreting HBsAg when compared to either a non-HBsAg producing epidermoid tumor or most other tissues obtained from nude mice treated with the 131I-labeled anti-Hbs McAb. A radiolabeled control McAb that did not recognize HBsAg failed to discriminate between either the HBsAg positive and negative tumors or other tissues from nude mice. These data demonstrate the in vivo immunological specificity of anti-HBs McAb for HBsAg associated with a hepatoma tumor.

Animals↗

PubMed1985

Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxyl-terminal fragment of human apolipoprotein B-100.

Apolipoprotein (apo) B-100 cDNAs were identified in a human liver cDNA library cloned in the expression vector lambda gt11. The beta-galactosidase-apoB-100 fusion protein was detected by two independently produced low density lipoprotein polyclonal antisera and by three apoB-100 monoclonal antibodies that crossreact with apoB-74. It was not recognized by two apoB-100 monoclonal antibodies that crossreact with apoB-26. The longest clone, lambda B8, was completely sequenced. It contains a 2.8-kilobase DNA fragment containing the codons for the carboxyl-terminal 836 amino acid residues of apo-B-100, as well as the 3' untranslated region of apoB-100 mRNA. We have thus mapped apoB-74 to the carboxyl-terminal portion of apoB-100. The deduced amino acid sequence of the cloned DNA matches the sequences of 14 apoB-100 peptides determined in our laboratory. Minor differences in amino acid sequence were noted in three of the peptides, suggesting polymorphism of apoB-100 at the protein and DNA levels. Secondary structure predictions reveal an unusual pattern for apolipoproteins, consisting of beta-structure (24%), alpha-helical content (33%), and random structure (30%). Ten amphipathic helical regions of 10-24 residues were identified. This carboxyl-terminal fragment of apoB-100 is considerably more hydrophobic than other apolipoproteins with known structure. Its lipid binding regions might include stretches of highly hydrophobic beta-sheets as well as amphipathic helices. Our findings on apoB structure might be important for understanding the role of apoB-100-containing lipoproteins in atherosclerosis.

Amino Acid Sequence↗

PubMed1985

Recognition of mos-related proteins with an antiserum to a peptide of the v-mos gene product.

A mos-specific antiserum was generated by injection of rabbits with a peptide predicted from the sequence of the v-mos gene of Moloney murine sarcoma virus (MuSV) strain 124. The peptide is composed of amino acids 37-55 (cyclized at the cysteine residues) conjugated to keyhole limpet haemocyanin. This serum [anti-mos(37-55c)] specifically recognized p37mos in MuSV-124 acutely infected NIH-3T3 cells, P85gag-mos in 6m2 cells, an NRK clone infected with the temperature-sensitive mutant (ts110) of Moloney MuSV, and P100gag-mos in 54-5A4 cells, an NRK clone infected with a spontaneous revertant of ts110. An additional protein of Mr 55000 from uninfected cells was recognized by this serum. Reactivity of the serum toward the v-mos-containing proteins and the 55K protein was completely inhibited by prior incubation with free peptide. The 55K protein was not recognized by antisera made from synthetic peptides prepared from the C-terminal eight or 12 amino acids of v-mos.

Animals↗

PubMed1985

Selective labelling of apolipoproteins A-I and C-I at methionine residues by (3H) methyl exchange.

Apolipoproteins C-I and A-I were radioactively labelled with tritium by (3H)-methyl exchange. The methionine residues were first methylated with (3H)-methyl iodide at pH 4 and the reaction products were purified by gel filtration and cation exchange chromatography. The products were then demethylated with 2-mercaptoethanol (6 M) at pH 8.6 to regenerate the apolipoproteins in an unmodified but tritiated form. The specific radioactivity for apolipoprotein C-I and A-I was 3.5 X 10(6) and 1.5 X 10(7) dpm/pmol respectively. The properties of (3H)-apolipoprotein C-I were examined by reversed phase HPLC and by incorporation into very low density lipoproteins (VLDL).

Apolipoprotein A-I↗

PubMed1985

Synthesis of peptide labelled with p-iodophenylalanine which corresponds to the first amphipathic region of apolipoprotein C-I.

The amino terminal fragment (1-15) of apolipoprotein C-1, H-Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe14-Gly was prepared by the solid phase method. However, the phenylalanine residue at position 14 was replaced with p-iodophenylalanine in the chemical synthesis. The preparation of t-butyloxy-carbonyl-p-iodophenylalanine is described. After completion of the synthesis, the product was deprotected and cleaved from the resin with liquid HF. The peptide was purified by gel filtration on Sephadex G-25 and preparative high performance liquid chromatography. The purified material was shown to be homogeneous by amino acid analytical data and by chromatography in three different analytical reversed phase HPLC systems. The peptide was then labelled by the chloroamine-T procedure and good incorporation of 125I was obtained. After purification of the product by gel filtration on Biogel P-2, the labelled pentadecapeptide was tested for ability to bind to Very Low Density Lipoproteins (VLDL) in the following manner: VLDL was isolated from normolipemic serum by ultracentrifugal flotation and 100-microliter samples were incubated with labelled material dissolved in 200 microliter of 0.5 M NaCl, 0.02 M sodium phosphate buffer, pH 7.4 at 37 degrees for 30 min. The VLDL fraction was again isolated by ultracentrifugal flotation and the incorporation of radioactivity into the lipoprotein was measured. Under these conditions, a sample of [3H]-native apolipoprotein C-I was incorporated to an extent of 83% of the total sample, while the [125I]-pentadecapeptide exhibited an incorporation of 8.7%.

Amino Acid Sequence↗

PubMed1985

Fluorine-, pyrene-, and nitroxide-labeled sphingomyelin: semi-synthesis and thermotropic properties.

A rapid, high-yield method has been developed for the N-acylation of sphingosine-1-phosphocholine (SPC) to obtain a series of sphingomyelin (SM) derivatives bearing different reporter groups in the N-acyl chain. The procedure utilizes a fatty acid activated as the N-hydroxysuccinimide ester. A 1:1 molar mixture of the activated fatty acid and SPC is refluxed in 5% aqueous NaHCO3-ethanol 9:1 (v/v) for 2-3 hr. After acidification, the precipitated SM is purified by column chromatography over silica gel. This procedure offers significant advantages over those reported for the synthesis of well-defined SM: i) only the amino (not the hydroxyl) group is acylated; ii) only one equivalent of fatty acid is required; and iii) the time necessary for the reaction to go to completion is short. The transition temperature and enthalpy of each SM derivative has been measured by differential scanning calorimetry and compared to its unlabeled analog.

Calorimetry, Differential Scanning↗

PubMed1984

In vitro binding of synthetic acylated lipid-associating peptides to high-density lipoproteins: effect of hydrophobicity.

To measure the effect of hydrophobicity on the binding of model apoproteins to lipoproteins, we synthesized a 15 amino acid lipid-associating peptide (LAP) with acyl chains of various lengths (0-18 carbons) bound to the N-terminal amino acid through a peptide bond. The acylated LAPs preferentially bound to high-density lipoprotein (HDL) and were activators of lecithin:cholesterol acyltransferase. Circular dichroic spectra indicated that the LAP association with phospholipid was accompanied by increased alpha-helical structure. The LAPs self-associated in solution as judged from tryptophan fluorescence analysis. These characteristics, which are comparable to those of apolipoprotein A-I, were strongly dependent upon the acyl chain length of the LAPs. The equilibrium constants (Keq) for the association of LAPs to reassembled HDL were measured by equilibrium dialysis at several temperatures. At 37 degrees C, Keq increased by 3 orders of magnitude as the number of carbon units was increased from 0 to 16; there was a log-linear relationship between Keq and the acyl chain length. The free energy of association (delta Ga) decreased by a constant value for each methylene unit added to the acyl chain (0.35 kcal mol-1), clearly demonstrating a strict hydrophobic effect. This change of delta Ga was enthalpy rather than entropy driven. Our data show that, with all other parameters including putative alpha-helicity, sequence, and molecular weight being constant, the binding of a lipid-associating peptide to lipoprotein is governed by its hydrophobicity.

Apolipoprotein A-I↗

PubMed1984

Enhancement of viral hepatitis B antibody (Anti-HBs) response to a synthetic cyclic peptide by priming with anti-idiotype antibodies.

In vivo injections of anti-idiotype antibodies were used to prime the immune system of mice to hepatitis B surface antigen (HBsAg). Anti-idiotype reagents in conjunction with a cyclic synthetic peptide analogous to positions 122-137 of HBsAg induced an antibody response to HBsAg (anti-HBs) comparable to that obtained with a single injection of intact HBsAg particles. In addition, high anti-HBs titers were produced in mice injected with HBsAg following anti-idiotype priming. These data indicate that anti-idiotype antibodies may be useful in priming the immune system of a host to a potential infectious agent.

Animals↗