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• W2000754433 abstract "The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1. The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1. HDL plays a central role in reverse cholesterol transport of excessively accumulated cholesterol from peripheral tissues to the liver (1Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 2Sviridov D. Nestel P. Dynamics of reverse cholesterol transport: protection against atherosclerosis.Atherosclerosis. 2002; 161: 245-254Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 3von Eckardstein A. Nofer J.R. Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 13-27Crossref PubMed Scopus (645) Google Scholar). The first and most important step in reverse cholesterol transport is a reaction in which lipid-free apoA-1, composed of only a single apoA-1, or lipid-poor apoA-1, composed of a single apoA-1 and a small amount of phospholipid, accepts unesterified cholesterol from cells through ATP binding cassette transporter A1 (ABCA1) on the plasma membrane to form a discoidal nascent HDL composed of two molecules of apoA-1, phospholipid, and unesterified cholesterol (4Yokoyama S. Assembly of high-density lipoprotein.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 20-27Crossref PubMed Scopus (106) Google Scholar, 5Rye K.A. Barter P.J. Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 421-428Crossref PubMed Scopus (262) Google Scholar). However, the presence or absence of lipid-free- or -poor apoA-1 in plasma and its composition and structure remain obscure (5Rye K.A. Barter P.J. Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 421-428Crossref PubMed Scopus (262) Google Scholar). Preβ1-HDL is an apoA-1-containing particle known as the initial plasma acceptor of cellular cholesterol (1Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 6Barrans A. Jaspard B. Barbaras R. Chap H. Perret B. Collet X. Pre-beta HDL: structure and metabolism.Biochim. Biophys. Acta. 1996; 1300: 73-85Crossref PubMed Scopus (88) Google Scholar, 7Wróblewska M. The origin and metabolism of a nascent pre-β high density lipoprotein involved in cellular cholesterol efflux.Acta Biochim. Pol. 2011; 58: 275-285Crossref PubMed Scopus (24) Google Scholar, 8Castro G.R. Fielding C.J. Early incorporation of cell-derived cholesterol into pre-beta-migrating high density lipoprotein.Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar). Although preβ1-HDL accounts for only 1–5% of total apoA-1 in plasma, it is considered to play an important role in cholesterol efflux because efflux capacity is remarkably reduced by adding anti-preβ1-HDL-specific antibody to plasma or depleting preβ1-HDL from plasma in cholesterol efflux experiments where plasma is added to culture cells (9Fielding P.E. Kawano M. Catapano A.L. Zoppo A. Marcovina S. Fielding C.J. Unique epitope of apolipoprotein A-I expressed in pre-beta-1 high-density lipoprotein and its role in the catalyzed efflux of cellular cholesterol.Biochemistry. 1994; 33: 6981-6985Crossref PubMed Scopus (85) Google Scholar, 10Kawano M. Miida T. Fielding C.J. Fielding P.E. Quantitation of pre beta-HDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid.Biochemistry. 1993; 32: 5025-5028Crossref PubMed Scopus (113) Google Scholar). Plasma preβ1-HDL can be separated by nondenaturing two-dimensional gel electrophoresis (2D electrophoresis). Plasma preβ1-HDL migrates to the preβ position in agarose gel electrophoresis for the first dimension, and to the position of the smallest particle among plasma apoA-1-containing particles in nondenaturing PAGE for the second dimension (6Barrans A. Jaspard B. Barbaras R. Chap H. Perret B. Collet X. Pre-beta HDL: structure and metabolism.Biochim. Biophys. Acta. 1996; 1300: 73-85Crossref PubMed Scopus (88) Google Scholar, 11Asztalos B.F. Sloop C.H. Wong L. Roheim P.S. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations.Biochim. Biophys. Acta. 1993; 1169: 291-300Crossref PubMed Scopus (216) Google Scholar). Preβ1-HDL is known to be present in the plasma of Tangier disease patients as the only apoA-1-containing particle (12Huang Y. von Eckardstein A. Shili W. Langer C. Assmann G. Generation of pre-beta 1-HDL and conversion into alpha-HDL. Evidence for disturbed HDL conversion in Tangier disease.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1746-1754Crossref PubMed Scopus (31) Google Scholar, 13Asztalos B.F. Brousseau M.E. McNamara J.R. Horvath K.V. Roheim P.S. Schaefer E.J. Subpopulations of high density lipoproteins in homozygous and heterozygous Tangier disease.Atherosclerosis. 2001; 156: 217-225Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Tangier disease is a disorder in which cholesterol efflux to apoA-1 is rendered defective by mutations in the ABCA1 gene (14Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. et al.Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1505) Google Scholar, 15Bodzioch M. Orsó E. Klucken J. Langmann T. Böttcher A. Diederich W. Drobnik W. Barlage S. Büchler C. Porsch-Ozcürümez M. et al.The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1345) Google Scholar, 16Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denèfle P. et al.Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1266) Google Scholar). In fibroblasts obtained from Tangier patients, cholesterol and phospholipid efflux to lipid-free apoA-1 is completely defective, and HDL is not formed (17Francis G.A. Knopp R.H. Oram J.F. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease.J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (373) Google Scholar). Hence plasma preβ1-HDL may be lipid-free or -poor apoA-1, the substrate (precursor) for ABCA1-mediated cholesterol efflux. However some reports claim that preβ1-HDL contains cholesterol (8Castro G.R. Fielding C.J. Early incorporation of cell-derived cholesterol into pre-beta-migrating high density lipoprotein.Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar, 18Guendouzi K. Jaspard B. Barbaras R. Motta C. Vieu C. Marcel Y. Chap H. Perret B. Collet X. Biochemical and physical properties of remnant-HDL2 and of pre beta 1-HDL produced by hepatic lipase.Biochemistry. 1999; 38: 2762-2768Crossref PubMed Scopus (33) Google Scholar, 19Jaspard B. Collet X. Barbaras R. Manent J. Vieu C. Parinaud J. Chap H. Perret B. Biochemical characterization of pre-beta 1 high-density lipoprotein from human ovarian follicular fluid: evidence for the presence of a lipid core.Biochemistry. 1996; 35: 1352-1357Crossref PubMed Scopus (62) Google Scholar), is composed of two molecules of apoA-1 (5Rye K.A. Barter P.J. Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 421-428Crossref PubMed Scopus (262) Google Scholar), and is a discoidal particle (2Sviridov D. Nestel P. Dynamics of reverse cholesterol transport: protection against atherosclerosis.Atherosclerosis. 2002; 161: 245-254Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 3von Eckardstein A. Nofer J.R. Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 13-27Crossref PubMed Scopus (645) Google Scholar). The reason the composition and structure of preβ1-HDL are still obscure may be that adequate preβ1-HDL for analysis cannot be easily purified from plasma. The concentration of plasma preβ1-HDL is much lower than that of other HDL subfractions, and preβ1-HDL is very unstable in plasma (20Miida T. Miyazaki O. Nakamura Y. Hirayama S. Hanyu O. Fukamachi I. Okada M. Analytical performance of a sandwich enzyme immunoassay for pre beta 1-HDL in stabilized plasma.J. Lipid Res. 2003; 44: 645-650Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We previously obtained a monoclonal antibody (MAb) 55201 (MAb55201) that specifically recognizes apoA-1 in preβ1-HDL and developed an ELISA for easily measuring plasma preβ1-HDL concentrations using the MAb (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). Recently a variety of research results on preβ1-HDL using the ELISA were reported, in particular the relationships between plasma preβ1-HDL concentrations and coronary artery disease and medications have been noted (22Sethi A.A. Sampson M. Warnick R. Muniz N. Vaisman B. Nordestgaad B.G. Tybjaerg-Hansen A. Remaley A.T. High pre-beta1 HDL concentrations and low lecithin:cholesterol acyltransferase activities are strong positive risk markers for ischemic heart disease and independent of HDL-cholesterol.Clin. Chem. 2010; 56: 1128-1137Crossref PubMed Scopus (75) Google Scholar, 23Tashiro J. Miyazaki O. Nakamura Y. Miyazaki A. Fukamachi I. Bujo H. Saito Y. Plasma pre beta1-HDL level is elevated in unstable angina pectoris.Atherosclerosis. 2009; 204: 595-600Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 24Troutt J.S. Alborn W.E. Mosior M.K. Dai J. Murphy A.T. Beyer T.P. Zhang Y. Cao G. Konrad R.J. An apolipoprotein A-I mimetic dose-dependently increases the formation of prebeta1 HDL in human plasma.J. Lipid Res. 2008; 49: 581-587Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 25Kawano M. Nagasaka S. Yagyu H. Ishibashi S. Pitavastatin decreases plasma prebeta1-HDL concentration and might promote its disappearance rate in hypercholesterolemic patients.J. Atheroscler. Thromb. 2008; 15: 41-46Crossref PubMed Scopus (28) Google Scholar, 26Miida T. Seino U. Miyazaki O. Hanyu O. Hirayama S. Saito T. Ishikawa Y. Akamatsu S. Nakano T. Nakajima K. et al.Probucol markedly reduces HDL phospholipids and elevated prebeta1-HDL without delayed conversion into alpha-migrating HDL: putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling.Atherosclerosis. 2008; 200: 329-335Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The aim of the present study is to clarify the composition and structure of plasma preβ1-HDL. The MAb55201 we obtained reacts with apoA-1 in plasma preβ1-HDL and delipidated apoA-1 (lipid-free apoA-1), and does not react with apoA-1 in other HDL sub-fractions (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). We first purified preβ1-HDL from fresh plasma of healthy subjects with affinity chromatography using MAb55201, and with gel filtration chromatography to analyze the protein and lipid composition and to examine its shape under electron microscopy. We then analyzed intact plasma preβ1-HDL and lipid-free apoA-1 by nondenaturing PAGE, nondenaturing 2D electrophoresis, and size-exclusion chromatography, and examined whether the concentration of lipid-free apoA-1 added to preβ1-HDL-depleted plasma decreases in an LCAT-dependent conversion into α-HDL, which is a phenomenon unique to plasma preβ1-HDL, for comparison of intact plasma preβ1-HDL and lipid-free apoA-1. In addition, we obtained another anti-preβ1-HDL MAb (MAb55205), which recognizes a different epitope on apoA-1 from that of MAb55201, and investigated whether the apoA-1 in preβ1-HDL is a monomer or dimer by an immunochemical procedure using the anti-preβ1-HDL antibodies. Blood was obtained on the day experiments were performed from healthy volunteers (employees of Sekisui Medical Co., Ltd.) having a normal plasma lipid profile (total cholesterol, <220 mg/dl; triglyceride, <150 mg/dl; and HDL-cholesterol, >40 mg/dl). Written informed consent was obtained from all volunteers. The subjects fasted overnight, and venous blood was drawn into vacuum blood collection tubes containing EDTA-2Na (Terumo Corporation, Tokyo, Japan). The blood samples were immediately chilled in ice water for 30 min and centrifuged at 2°C, 3,000 rpm for 30 min to separate the plasma. Lipid-free apoA-1 was prepared by delipidating the HDL fraction (1.063 g/ml < d < 1.21 g/ml) and subsequent purification as described previously (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). Lipid-free apoA-1 is known to self-associate to form dimers and larger multimers, depending on various conditions that include apoA-1 concentration and ionic strength in the solution (27Formisano S. Brewer Jr, H.B. Osborne Jr, J.C. Effect of pressure and ionic strength on the self-association of apo-A-I from the human high density lipoprotein complex.J. Biol. Chem. 1978; 253: 354-359Abstract Full Text PDF PubMed Google Scholar). The purified lipid-free apoA-1 was dialyzed against PBS [0.15 mol/l NaCl, 20 mmol/l phosphate buffer (pH 7.2)] to a concentration of 0.1 mg/ml or lower, and stored at −80°C to avoid self-association before use. Twenty-five peptides of apoA-1 were purchased from Protein Purity Ltd. (Gunma, Japan). The molecular mass of each peptide was confirmed to be the same as the theoretical molecular mass by MALDI-MS, and the purity was confirmed to be more than 95% by HPLC. Cyanogen bromide (CNBr) fragments of apoA-1 were prepared using the procedure reported by Morrison et al. (28Morrison J. Fidge N.H. Tozuka M. Determination of the structural domain of apoA1 recognized by high density lipoprotein receptors.J. Biol. Chem. 1991; 266: 18780-18785Abstract Full Text PDF PubMed Google Scholar) as follows. Lipid-free apoA-1 (0.1 mg) dialyzed with purified water was lyophilized and dissolved in 0.2 ml of 70% trifluoroacetic acid (TFA). CNBr dissolved in 0.2 ml of 70% TFA to a concentration of 1 mg/ml was added to the apoA-1 solution; the bottle was suffused with nitrogen gas and sealed, followed by incubation at room temperature for 24 h in the dark. After incubation, 3 ml of purified water was added and lyophilized. Each fragment was identified by molecular mass using SDS-PAGE and N-terminal amino acid analysis. MAb55201 was obtained by our group as described previously (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). The mouse anti-influenza A MAb obtained by our group was used as the control IgG. MAb55201 or the control IgG-bound sepharose column was prepared by using CNBr-activated Sepharose 4B (GE Healthcare) in accordance with the procedure recommended by the manufacturer. Polyacrylamide slab gels used for nondenaturing PAGE or SDS-PAGE, and the protein transfer kits used for Western blotting were purchased from Cosmo Bio Co., Ltd. (Tokyo). DTNB used for the LCAT-dependent conversion experiment was purchased from Sigma-Aldrich, and dissolved with the phosphate buffer [0.1 mol/l KH2PO4-Na2HPO4 (pH 7.4)] just prior to use, followed by addition to plasma or preβ1-HDL-depleted plasma to a final concentration of 2 mmol/l. Goat anti-apoA-1 polyclonal antibody (PAb) and goat anti-apoA-2 PAb were prepared as described previously (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). Biotin-labeled antibodies were prepared with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Inc.). Plasma (40 ml) was passed through the control IgG-bound Sepharose column (5 ml) to remove fractions nonspecifically binding to mouse IgG or the Sepharose beads. The eluent (40 ml) was then passed through the MAb55201-bound Sepharose column (2 ml) to bind preβ1-HDL. The unbound fraction was used as the preβ1-HDL-depleted plasma for the experiments described later. After washing the MAb55201-bound Sepharose column with 30 ml of PBS, the adsorbed fraction was eluted with 10 ml of 0.1 mol/l glycine-HCl buffer (pH 2.0) containing 0.15 mol/l NaCl, and collected in 1 ml fractions. The absorbance at 280 nm of each fraction was measured, and the fractions (Fr. 4–6) having absorbance of more than 0.4 were immediately pooled and applied to a Superdex200 16/60 column (GE Healthcare) for further separation by gel filtration chromatography. The chromatography was run with 0.15 mol/l NaCl solution at a flow rate of 1.0 ml/min and the eluent was collected in 1.0 ml fractions by a fraction collector. The preβ1-HDL concentration of each fraction was determined by ELISA using MAb55201 (preβ1-HDL-ELISA) (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). Those fractions containing a high concentration of preβ1-HDL were then collected. The purification process was performed four times, once each for the plasma samples obtained from the four healthy subjects, and all operations were performed at 4°C. The purity of preβ1-HDL in the purified sample was confirmed by nondenaturing 2D electrophoresis, and the concentration of preβ1-HDL was determined by preβ1-HDL-ELISA. In order to examine the possibility that MAb55201 bound to Sepharose beads may strip apoA-1 from HDL, we carried out affinity chromatography under the same conditions at 1/10 scale of the above using HDL (1.063 g/ml < d < 1.21 g/ml) prepared by ultracentrifugation instead of plasma. Two milliliters of HDL (140 μg/ml of cholesterol) was applied to a MAb55201-bound Sepharose column (0.2 ml). After washing the column with 3 ml of PBS, the adsorbed fraction was eluted with 1 ml of glycine-HCl buffer (pH 2.0). The eluted fraction was neutralized by adding 0.05 ml of 2 mol/l Tris-HCl buffer (pH 8.0), followed by analysis by nondenaturing PAGE. Purified preβ1-HDL (4 μl) or plasma (2 μl) was added to a 0.75% agarose gel in 50 mmol/l Barbital buffer (pH 8.6) prepared on gel-bond film with a thickness of 1 mm, and run at 540 V for 60 min at 4°C. Strips of agarose gel were cut out, set on 10–20% polyacrylamide gradient slab gels, and run at 75 V for 24 h at 4°C in 90 mmol/l Tris, 80 mmol/l boric acid, and 3 mmol/l EDTA (pH 8.3). The separated HDL subfractions in the polyacrylamide gel were transferred onto poly vinylidene difluoride (PVDF) membranes using a semidry electroblotter, and then analyzed by Western blot using goat anti-apoA-1 PAb, HRP-conjugated rabbit anti-goat IgG antibody (DAKO), and diaminobenzidine. Purified preβ1-HDL solution was dropped on a 400 mesh grid coated with carbon film for dispersion. The adsorbed specimen was soaked with 2% uranyl acetate at 4°C for 10 s for negative staining and examined in a transmission electron microscope, JEM-1200EX (JEOL Ltd., Tokyo, Japan). The protein content was determined with a Micro BCA kit (Thermo). The protein composition was investigated by SDS-PAGE and mass spectrometric analysis as follows. The purified fraction (0.22 μg) diluted in a sample buffer containing SDS and 2-mercaptoethanol was boiled and electrophoresed on a 15% polyacrylamide gel. Mass spectrometric analysis and the pretreatment were performed as described previously (29Mori T. Kitani Y. Ogihara J. Sugiyama M. Yamamoto G. Kishida O. Nishimura K. Histological and MS spectrometric analyses of the modified tissue of bulgy form tadpoles induced by salamander predation.Biol. Open. 2012; 1: 308-317Crossref PubMed Scopus (7) Google Scholar). Briefly, protein in the gel was detected by MS-compatible silver staining. The stained band was cut out and put into a 1.5 ml tube. Cysteine disulfide bonds were reduced with DTT and alkylated with iodoacetamide. In-gel digestion with trypsin was performed at 37°C overnight, followed by extracting the peptides with 50% acetonitrile containing 5% TFA. The extracts were dried and redissolved in 0.1% formic acid, followed by LC/MS/MS analysis. LC/MS/MS analysis was carried out in a LCQ Deca XP ion trap mass spectrometer (ThermoFinnigan, USA) equipped with a nano-LC electrospray ionization source (AMR, Japan), interfaced on-line with a capillary HPLC system (Paradigm MS4; Michrom BioResources, USA). MS/MS data were analyzed using SEQUEST, a computer program that allows the correlation of experimental data with theoretical spectra generated from known protein sequences, to be compared against the latest version of the public nonredundant protein database of the National Center for Biotechnology Information. The amounts of choline-containing phospholipids, cholesterol, and triglyceride were determined by enzymatic methods using commercial reagents (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as follows. Each standard, diluted with saline (0.15 mol/l NaCl) and purified preβ1-HDL (0.2 ml), were added to the appropriate enzyme solution (0.2 ml) in glass tubes and incubated at 37°C for 5 min. Absorbance at 600 nm was measured by a spectrophotometer, UV-2400PC (Shimadzu Corporation, Kyoto, Japan). In order to validate each lipid assay, limit of detection (LOD) and limit of quantification (LOQ) were calculated using the following equations, in accordance with the appropriate ICH guideline (30.ICH Harmonised Tripartite Guideline. 2005. Validation of analytical procedures: text and methodology Q2(R1). Proceedings of the International Conference on Hormonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use in London, UK.Google Scholar). LOD=3.3σ/S LOQ=10σ/S where σ and S represent standard deviations of blank absorbance (n = 10) and slopes of calibration curves, respectively. Plasma and preβ1-HDL-depleted plasma were diluted 10-fold with sample buffer containing 31% sucrose, 0.06% EDTA, and 0.01% Bromophenol blue; and purified preβ1-HDL and lipid-free apoA-1 were diluted with the sample buffer to a concentration of 10 μg/ml. The unbound fraction and the eluted fraction in affinity chromatography using HDL were each diluted 2-fold with the sample buffer. Each sample was applied to a 15–25% polyacrylamide gradient slab gel with 5 μl/well and electrophoresed at 75 V for 24 h at 4°C. Then Western blot analysis was performed in the same manner as described above for nondenaturing 2D electrophoresis. A high molecular mass calibration kit for native electrophoresis (GE Healthcare) was used as the molecular size marker. Plasma, preβ1-HDL-depleted plasma, and lipid-free apoA-1 added to preβ1-HDL-depleted plasma were individually separated using size-exclusion chromatography in accordance with the methods of Nanjee and Brinton (31Nanjee M.N. Brinton E.A. Very small apolipoprotein A-I-containing particles from human plasma: isolation and quantification by high-performance size-exclusion chromatography.Clin. Chem. 2000; 46: 207-223Crossref PubMed Scopus (53) Google Scholar). Fifty microliters of each sample was applied to a Superdex 200 HR 10/30 column connected in series to a Superdex 75 HR 10/30 column and separated in a 50 mmol/l Tris-HCl buffer (pH 7.4), containing 150 mmol/l NaCl, 1 g/l sodium EDTA, and 1 g/l NaN3 at a flow rate of 0.25 ml/min. After discarding 12.5 ml of the eluent, 40 fractions of 0.5 ml each were collected. The concentrations of preβ1-HDL and lipid-free apoA-1 in all fractions were determined by preβ1-HDL-ELISA (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). The apoA-1 concentrations of fractions separated from plasma were determined by sandwich ELISA using goat anti-apoA-1 PAb as described previously (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). Plasma and lipid-free apoA-1 added to preβ1-HDL-depleted plasma (0.5 ml) were individually incubated at 37°C with or without DTNB. A portion of each sample was taken at intervals and diluted 11-fold with a stabilization buffer (20Miida T. Miyazaki O. Nakamura Y. Hirayama S. Hanyu O. Fukamachi I. Okada M. Analytical performance of a sandwich enzyme immunoassay for pre beta 1-HDL in stabilized plasma.J. Lipid Res. 2003; 44: 645-650Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) containing 50% sucrose. The diluted samples were immediately stored at −80°C, and later the concentrations of preβ1-HDL and lipid-free apoA-1 were determined by preβ1-HDL-ELISA (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). A hybridoma cell line producing MAb55205 was developed in the same manner as described previously for MAb55201 (21Miyazaki O. Kobayashi J. Fukamachi I. Miida T. Bujo H. Saito Y. A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels.J. Lipid Res. 2000; 41: 2083-2088Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, after immunization of a Balb/c mouse with lipid-free apoA-1, the mouse was euthanized, and the spleen cells were fused with murine myeloma cells according to the method of Kohler and Milstein (32Köhler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.Nature. 1975; 256: 495-497Crossref PubMed Scopus (12712) Google Scholar). Screening of hybridoma cells was performed using two kinds of ELISA with biotin-labeled goat anti-apoA-1 or apoA-2 PAb. Hybridomas were selected that produced antibodies showing reactivity in ELISA using anti-apoA-1 PAb, but not in ELISA using anti-apoA-2 PAb. A hybridoma cell line producing anti-apoA-1 MAb 14208 (MAb14208) was prepared as follows. After immunization and cell fusion were performed as described above, a hybridoma cell producing an antibody that reacted strongly with apoA-1 in apoA-1-immobilized ELISA was selected. The selected hybridomas were cloned by limited dilutio" @default.
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• W2000754433 date "2014-02-01" @default.
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• W2000754433 cites W2062072221 @default.
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