1,723,257 research outputs found
Cross reactivity characterisation of anti-CS-6253 antibody.
No full text<p>Non denaturing gradient gel electrophoresis (4–20%) is used to separate CS-6253 complexes, apo A-I and apo E. CS-6253 was immunoblotted using antibody against human apo A-I, human apo E and against CS-6253. Normolipidemic human plasma was used as positive control. Molecular weight markers were revealed by Ponceau S. Left panel shows dose response relationship with an antibody directed against CS-6253. Middle panel shows anti-apo A-I antibody does not cross-react with CS-6253; similarly, right panel shows antibody against apo E does not react against CS-6253.</p
Carolingian Critters III: Munich, BSB Clm 6253
No full textThe pick of today is slightly less Carolingian than the other critters that were paraded here. It is another manuscript from Bayerische Staatsbibliothek in Munich with a shelfmark Clm 6253. Munich, BSB, Clm 6253, fol. 243r Clm 6253, the first volume of a three-volume copy of Expositio Psalmorum of Cassiodorus[1], was produced at Freising in the second quarter of the ninth century, during the times of Hitto (811/812-836) and his nephew Erchenbert (836-854).[2] This period saw copying of as ma..
Time course ability of CS-6253 to generate nHDL-mimetic like particles.
No full text<p>(A and B): nHDL-CS-6253 and nHDL-apo A-I released to the medium at specific time points (45 min and 6 h) from BHK cells expressing ABCA1 were analyzed by 2D-PAGGE. Apo A-I was detected by polyclonal anti-apo A-I, CS-6253 peptides was detected by anti-CS-6253 and revealed by chemiluminescence. Molecular markers are indicated.</p
Dynamics of transfer and redistribution of BHK-ABCA1 cell-derived cholesterol content of nHDL-CS-6253.
No full text<p>nHDL-CS-6253 particles were labelled with cell derived <sup>3</sup>[H]cholesterol and incubated with normolipidemic plasma for various time periods at 37°C (1μg peptide: 10μg plasma apo A-I). A. Characterization of lipid particles released to the medium in the presence of CS-6253. <sup>3</sup>[H]-cholesterol-labelled BHK cells expressing ABCA1 or not were incubated in the presence of 0.96 μM apo A-I (solid line) or CS-6253 (dashed line) for 45min at 37°C. Concentrated medium from cells was analysed by FPLC, and radioactivity associated with each fraction was determined. B. Transfer of total <sup>3</sup>[H]cholesterol from <sup>3</sup>[H]nHDL-CS-6253 to plasma lipoproteins. nHDL-CS-6253 or nHDL-apo A-I were generated after incubation (4h) of BHK cells expressing ABCA1 with increased doses of lipid free CS-6253 or apo A-I. Media cell culture containing <sup>3</sup>[H]-nHDL-particles was incubated in human plasma for 1h, 37°C. After ApoB precipitation, fractions were dialysed and counted for radioactivity. Kinetic parameters for cholesterol transfer to apoB particles in plasma: nHDL-CS-6253 (439±0.20μM), <i>V<sub>max</sub></i> (439± 19.74%cpm/μl) vs nHDL-apo A-I (0.37 ± 0.035), <i>V<sub>max</sub></i> (517±24.45).C. Effects of <i>ex vivo</i> incubation of CS-6253 on plasma cholesterol lipoproteins. FPLC cholesterol profiles (OD 490 nm) of plasma apo A-I: peptide ratio (1:1) (dashed line) and plasma apo A-I: peptide ratios (1:0; i.e. apo A-I alone) (solid line) are obtained after 5 min incubation at 37°C. In all experiment counts were made in triplicate, (*P<0.05 by Student‘s t-test). For reference purposes, the <i>inset</i> shows an FPLC profile of normal plasma. D. Effect of <i>ex vivo</i> incubation of CS-6253 on plasma lipoproteins after separation by ND-PAGGE (5–35%) electrophoresis. CS-6253 (0.96 μM final dose) was incubated for 1h at 37°C with HDL, LDL or VLDL isolated by ultracentrifugation or with human plasma and then separated by electrophoresis at concentration of 1 mg/protein/mL. Gel is followed by Western blot analysis with anti-CS-6253 antibody. Internal control was run on the left of the gel and detected by Ponceau S.</p
Effect of the ABCA1 agonist CS-6253 on amyloid-beta and lipoprotein metabolism in cynomolgus monkeys
Get PDFBACKGROUND: Inducing brain ATP-binding cassette 1 (ABCA1) activity in Alzheimer's disease (AD) mouse models is associated with improvement in AD pathology. The purpose of this study was to investigate the effects of the ABCA1 agonist peptide CS-6253 on amyloid-β peptides (Aβ) and lipoproteins in plasma and cerebrospinal fluid (CSF) of cynomolgus monkeys, a species with amyloid and lipoprotein metabolism similar to humans. METHODS: CS-6253 peptide was injected intravenously into cynomolgus monkeys at various doses in three different studies. Plasma and CSF samples were collected at several time points before and after treatment. Levels of cholesterol, triglyceride (TG), lipoprotein particles, apolipoproteins, and Aβ were measured using ELISA, ion-mobility analysis, and asymmetric-flow field-flow fractionation (AF4). The relationship between the change in levels of these biomarkers was analyzed using multiple linear regression models and linear mixed-effects models. RESULTS: Following CS-6253 intravenous injection, within minutes, small plasma high-density lipoprotein (HDL) particles were increased. In two independent experiments, plasma TG, apolipoprotein E (apoE), and Aβ42/40 ratio were transiently increased following CS-6253 intravenous injection. This change was associated with a non-significant decrease in CSF Aβ42. Both plasma total cholesterol and HDL-cholesterol levels were reduced following treatment. AF4 fractionation revealed that CS-6253 treatment displaced apoE from HDL to intermediate-density- and low density-lipoprotein (IDL/LDL)-sized particles in plasma. In contrast to plasma, CS-6253 had no effect on the assessed CSF apolipoproteins or lipids. CONCLUSIONS: Treatment with the ABCA1 agonist CS-6253 appears to favor Aβ clearance from the brain
Effect of CS-6253 treatment on cholesterol efflux.
No full text<p>A. Time-course activity of ABCA1-mediated cholesterol efflux to lipid-free CS-6253 (30 μg/ml) and apo A-I (10 μg/ml), using BHK cells expressing ABCA1, and control cells (unstimulated BHK-ABCA1 and BHK-mock cells). Cholesterol efflux from either ABCA1 cells or control cells to apo A-I (closed circles), and CS-6253 (closed triangles) was determined at the indicated time points. B. Dose dependent ABCA1 mediated cholesterol efflux in BHK-ABCA1 induced with mifepristone. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK-ABCA1 cells to apo A-I: <i>K<sub>m</sub></i> = 4.53±0.67 μg/ml (0.15±0.02 μM), <i>V<sub>max</sub></i> = 14.85±0.02% efflux/4h, and relative catalytic efficiency: <i>V<sub>max</sub>/K<sub>m</sub></i> = 3.27. CS-6253: <i>K<sub>m</sub></i> = 2.27±0.16 μg/ml (0.73±0.05 μM), <i>V<sub>max</sub></i> = 15.25±0.05% efflux/4h, and relative catalytic efficiency: <i>V<sub>max</sub>/K<sub>m</sub></i> = 6.71. ATI-5261: <i>K<sub>m</sub></i> = 1.04 ± 0.16 μg/ml (0.37 ± 0.04 μM), <i>V<sub>max</sub></i> = 14.48 ± 0.29% efflux/ h, and relative catalytic efficiency: <i>V<sub>max</sub>/K<sub>m</sub></i> = 13.92. Apo E: <i>K<sub>m</sub></i> = 10.30±2.84 μg/ml (0.21±0.05 μM), <i>V<sub>max</sub></i> = 11.45±1.16% efflux/4h, and relative catalytic efficiency: <i>V<sub>max</sub>/K<sub>m</sub></i> = 1.11. C. Ability of CS-6253 to stimulate cholesterol efflux from human macrophages THP-1. Foam cells were incubated with CS-6253 or apoA-I at equimolar ratio (0.96 μM) for 4h. Cholesterol efflux induced by CS-6253 and apoA-I were compared to control cells incubated alone. P<0.001 by Student’s t-test. D. Membrane ABCA1 levels assessment by Western blotting assays. ABCA1 was detected from THP-1 foam cells lysis at 4°C with lysis buffer containing 0.5% n-dodecylmaltoside in the presence of a protease inhibitor mixture followed by low speed centrifugation to remove cell debris. Protein concentration was determined by standard assay (Bio-Rad). The supernatants were then separated by SDS-PAGE (4–22.5%) in duplicate. After electrophoresis, ABCA1 was detected by an anti-ABCA1 antibody. GAPDH was used as loading control. E. Quantification of FC and CE in foam cells after interaction with CS-6253 and apoA-I. After a period of 4h cholesterol efflux, THP-1 foam cell lipids were extracted with hexane: isopropanol (3v/2v) as under ‘‘Material and Methods’. 3[H]-FC and 3[H]-CE were separated by TLC and located by exposure to iodine vapor, and were scraped off into liquid scintillating vials and assayed for radioactivity. Results are from a single experiment using triplicate wells and the mean (±SD) is presented. P<0.0001 versus control sample untreated THP-1 cells for cholesterol efflux. P<0.05 versus FC or CE in untreated cells, by Student’s t-test.</p
Dose dependent effects of pre-incubation of plasma with lipid free CS-6253, or nHDL-CS-6253 on ABCA1 cholesterol efflux.
No full text<p>A. Increased doses of lipid free CS-6253 or apo A-I were added to normolipidemic plasma and incubated for 1h at 37°C. A volume of 2.8% of HDL fractions isolated by PEG was used for the measurements of cholesterol efflux from BHK cells expressing ABCA1 and mock cells. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK expressing ABCA1 cells to apo A-I in plasma: <i>K<sub>m</sub></i> = 0.03±0.008 μM, <i>V<sub>max</sub></i> = 9.26±0.29% efflux/4h. CS-6253: <i>K<sub>m</sub></i> = 0.20±0.05 μM, <i>V<sub>max</sub></i> = 14.71±0.39% efflux/4h. Kinetic parameters for cholesterol efflux from BHK-mock cells to apo A-I in plasma: <i>K<sub>m</sub></i> = 0.07±0.95 μM, <i>V<sub>max</sub></i> = 0.30±0.15% efflux/6h. CS-6253: <i>K<sub>m</sub></i> = 0.65±0.63 μM, <i>V<sub>max</sub></i> = 0.84±0.17% efflux/4h. B. nHDL-CS-6253 and nHDL-apo A-I were generated as indicated above. nHDL particles were incubated in plasma for 1h at 37°C, and specific HDL fractions isolated by PEG were subjected to cholesterol efflux in <sup>3</sup>[H]-radiolabelled BHK cells. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK expressing ABCA1 cells to nHDL-apo A-I in plasma: <i>K<sub>m</sub></i> = 0.25±0.08 μM, <i>V<sub>max</sub></i> = 15.73±2.14% efflux/4h. nHDL-CS-6253: <i>K<sub>m</sub></i> = 0.32±0.15 μM, <i>V<sub>max</sub></i> = 15.01±0.84% efflux/4h. C. Mobilization of cholesterol efflux of increasing concentrations of peptide: plasma apo A-I molar ratios: (1:0) (closed circles), (1:10) (upright closed triangles), and (1:1) (closed squares). (C <i>inset</i>). The double reciprocal-plot was used to calculate the apparent appK<sub><i>m</i></sub> of the efflux activity in peptide: plasma with indicated ratios. Kinetics values are as follows, molar ratio of plasma apo A-I: CS-6253 (1:0); app<i>K<sub>m</sub></i> (28±0.07 μM). Molar ratio of plasma apo A-I: CS-6253 (1:1); app<i>K<sub>m</sub></i> (4.17±0.47 μM). Molar ratio of plasma apo A-I: CS-6253 (1:10); app<i>K<sub>m</sub></i> (0.71±0.46 μM). D. Lipid free CS-6253 was incubated with normolipidemic plasma, using peptide: apo A-I mole ratio of (1:1) for 5 min at 37°C versus plasma alone. Isolated HDL fractions by PEG were separated by 2D-PAGGE and HDL subpopulations were detected by apo A-I Western blot. Molecular size markers are shown. In all experiments, efflux of <sup>3</sup>[H]cholesterol is shown as means ± SD of triplicate experiments.</p
A proposed <i>in-vitro</i> model for lipid free CS-6253 lipidation and remodelling in RCT pathway.
No full text<p>1). ABCA1 interaction: a. ABCA1 oligomerization, b. Binding to ABCA-1. 2). Lipid efflux process: a. PM microdomains lipid desorption, b. ABCA1 and ABCG1 induction of lipid efflux and nHDL-CS-6253 formation, 3–4) HDL-CS-6253 remodelling by LCAT/PLTP in plasma, 5) generation of mature HDL-CS-6253, 6) Cholesterol uptake by liver via SR-BI, and 7) lipid transfer to both apoB-containing particles and the plasma HDL resident pool. 8) Although the mechanisms underlying this process is presently ambiguous, it is possible that nHDL-CS-6253 remodelling may lead to loss or ‘shedding’ of CS-6253 from HDL-CS-6253 in plasma as they are actively delipidated of phospholipids by PLTP to yield lipid poor apo A-I precursor of RCT activation. 9) Alternatively lipid free CS-6253 associates with αHDL-apo A-I species leading to preβ1-apo A-I formation, that can also close the loop and initiate again RCT. 10) in this process, the peptide transfer cholesterol to apoB particles, and 11) cholesterol is delivered to liver by the LDLR.</p
- …
