Lipid clearance assay and native gel analyses of rHDL formation.

<p>(A) ApoA-I WT and A164S were combined with DMPC at a 1:100 molar ratio and lipid binding measured by absorbance at 325nm at indicate times. Readings were fitted to one-way decay of non-linear regression. (B) Bar graph shows the lipid clearance rate described as t1/2 values calculated from panel A. Data are mean ±SEM (* = p<0.05, n = 3). (C) ApoAI-A164S and apoA-I WT were incubated with DMPC lipids at 37°C for indicated times and analyzed by native gel. <i>Arrows</i> indicate apoA-I WT and apoA-I A164S rHDL particles of diameters of approximately 10 nm. Lipid-free (LF) proteins at time 0 h prior to mixing with DMPC lipids are included as controls. (D) Formation of HDL upon incubation of apoA-I WT and A164S with rat serum. 0.75mg/ml of WT or A164S apoA-I was incubated with 200 μl rat serum. Samples were collected at indicated times and equal amounts of protein (0.56μg) separated by blue native PAGE and western blotting for human apoA-I was performed. Lipid-free (LF) proteins (not incubated with serum) and serum without protein (ctrl) are included as controls.</p

Cholesterol efflux from macrophages.

<p>J774 macrophages enriched with ³H-cholesterol were incubated with apoA-I WT or A164S and cholesterol efflux quantified as a function of protein concentration (A) or time (B) by scintillation counting of the resulting treatment media. An ACAT inhibitor and CPT-cAMP were used to prevent formation of cholesteryl esters of the ³H-cholesterol and to induce expression of ABCA1, respectively. Data from concentration gradient experiments were fitted using the Michaelis-Menten equation. Time dependent efflux treatments were performed with 50 μg/ml apoA-I WT or A164S. Each figure represents 3 independent experiments and displays mean±SD. (C) ApoA-I WT or A164S were incubated with rat serum for 2 h followed by incubation with J774 macrophages enriched with ³H-cholesterol for 2 h (10 μg/ml apoA-I). Rat serum was used as control for background efflux. Data is mean ±SEM (**** = p<0.0001, n = 3).</p

Limited proteolysis analysis.

<p>ApoA-I WT, A164S and L178H (5μg) were incubated for indicated times at 37°C in the presence of chymotrypsin. The cleaved products from limited proteolysis from different time points were separated by SDS-PAGE and visualized by coomassie staining. <i>Arrows</i> indicate migration distances of full-length proteins.</p

Analysis of alpha-helical content and thermal stability of apoA-I A164S.

<p>(A) CD analysis of apoA-I WT and A164S. Alpha-helical content of 0.2 mg/ml of apoA-I was calculated from molar ellipticity at 222 nm (n = 3). (B) Thermal stability of 0.2 mg/ml of apoA-I was assessed from normalized sigmoidal decrease of molar ellipticity at 222 nm. The results are mean ± SEM (n = 3). (C) Stopped-flow coupled to CD: apoA-I WT and A164S (0.5 mg/ml final concentration) were mixed with 100 mM SDS at the volume ratio 1:5 and molar ellipticity at 222 nm was measured. (D). Bar graph shows the t1/2 of apoA-I WT and A164S. Data is mean ±SEM (* = p<0.05, n = 6). (E) Near CD analysis of apoA-I WT and A164S (1.25 mg/ml).</p

Concentration dependences.

<p>The dependence of the peak H height on MT addition to the sample of human urine (A) and blood serum (B) of healthy people. In the inset pictures, the linear sections of studied dependences are shown. Chronopotentiogram demonstrates the signal 5 nM of MT after the addition into the human urine and signal of MT in blood serum.</p

Adsorptive transfer technique.

<p>The adsorptive transfer technique is based on the sample accumulation onto the working electrode surface and consequently on the electrode washing and measurement (A). CPSA scans of Cys, GSH, GSSG, PC2, BSA and MT in concentration of 100 nM (B). The supporting electrolyte was composed of 0.1 M H₃BO₃+0.05 M Na₂B₄O₇. AdTS CPSA parameters were as follows: starting potential 0 V, ending potential –1.85 V, temperature 20°C, time of accumulation 120 s; no reducing agent was added.</p

The affecting of peak H by various experimental conditions.

<p>The dependence of peak H on the temperature (A), different ionic strength (B), time of accumulation (C), osmolality (D) and concentration varying from 0 to 10 fmol (E) and from 0 to 320 amol (F). The concentration of MT was 5 fmol in 5 µl drop of sample. The signal height 40 000 (5 fmol) and 60 000 (500 fmol) s/V corresponds to 100%.</p

The effect of tris(2-carboxyethyl)phosphine (TCEP).

<p>Dependences of peak H height of Cys, GSH, GSSG, PC2, BSA (A) and its potential (B) on the different TCEP concentration; the picture in inset: chemical structure of TCEP. The influence of TCEP on CPSA signals of 40 fmol MT (5 µl, C). Dependences of peak H height and potential of 40 fmol MT (5 µl) on different TCEP concentration (D). The peak height 55 000 s/V corresponds to 100% (B).</p

Detection limits of thiols (<i>n</i> = 5) using AdTS CPSA method.

a<p>The linear section dependence of the studied thiol concentration.</p>b<p>The limit of Detection (3 S/N).</p>c<p>The limit of Quantification (10 S/N).</p><p>*Per drop sample (5 µl).</p

Cancer samples.

<p>The total amount of MT analysed in neuroblastoma cells sensitive (UKF-NB4) and resistant (UKF-B4) to cisplatin. SDS-PAGE band in MT position (300 ng for the control) and chronopotentiograms are displayed as the inset pictures (A). The MT chronopotentiograms of the human blood serum of the patients with the malignant breast tumour and MT amount of the individual patients (B).</p