BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIGS. 1A-C are graphs indicating the levels of paraoxonase activities in FPLC fractionated serum from healthy subjects. Serum samples (100 μl diluted X2) from 3 healthy subjects were fractionated by FPLC and sixty fractions of 0.5 ml each were collected and the protein absorbance was monitored at 260 nm (FIG. 1A), Arylesterase (FIG. 1B) and paraoxonase (FIG. 1C) activities were measured in all fractions. Results are given as mean±SD (n=3).
FIGS. 2A-C are bar graphs indicating the levels of paraoxonase activities in the HDL and in the LPDS fractions separated from the serum of healthy subjects by density gradient ultracentrifugation. The serum samples (3.5 ml) of 3 healthy subjects were fractionated and paraoxonase (FIG. 2A), arylesterase (FIG. 2B), and lactonase (FIG. 2C) activities were measured in the HDL and LPDS fractions. Results represent the mean±SD (n=3), and are given as units in fraction which originated from 1 ml of serum. *p<0.01 vs. HDL.
FIGS. 3A-D are bar graphs comparing HDL and LPDS PON1 paraoxonase activity and protein in fractions isolated from the serum of healthy subjects and diabetic patients. The HDL and LPDS fractions were isolated from the serum of 3 healthy subjects (Controls) or from 3 diabetic patients by FPLC or by density gradient ultracentrifugation (UC). Paraoxonase activity was determined in the FPLC HDL and LPDS fractions (FIG. 3A), as well as in the UC fractions (FIG. 3B). PON1 protein was analyzed by Western blot analysis, and the densitometric analysis of the protein bands, as well as the bands pictures are given for the FPLC (FIG. 3C) and UC (FIG. 3D) fractions. HDL-C: Control HDL, HDL-D: diabetic HDL, LPDS-C: Control LPDS, LPDS-D; Diabetic LPDS. Results are presented as mean±SD. #p<0.01 Diabetic HDL vs. Control HDL. *p<0.01 Diabetic LPDS vs. control LPDS.
FIGS. 4A-B are bar graphs illustrating the effect of HDL or LPDS enrichment with PON1 on their susceptibility to AAPH-induced lipids peroxidation. FIG. 4A: HDL and LPDS (5 paraoxonase units/ml) were incubated with evolved PON1 (50 paraoxonase U/ml), as well as with the PON1 vehicle solution for 2 hours at 37° C. The HDL and LPDS samples were then incubated with or without 100 mM AAPH for 2 hours at 37° C. The extent of AAPH-induced lipid peroxidation was measured by the lipid peroxides assay and calculated as described under the Methods section. FIG. 4B: HDL and LPDS (5 paraoxonase units/ml) were incubated with evolved PON1 (50 paraoxonase units/ml) for 2 hours at 37° C., and paraoxonase activity was determined at the end of the incubation period. The calculated values are the sum of the values obtained for HDL or LPDS alone+added PON1 activity. Results are given as mean±S.D of three different experiments. *p<0.01 vs. HDL.
FIG. 5 is a bar graph illustrating the difference in the ability of HDL and LPDS (derived from healthy subjects) to induce macrophage cholesterol efflux: effect of PON1 enrichment. HDL and LPDS were isolated from 3 healthy subjects by ultracentrifugation. J774 A.1 macrophages were and incubated at 37° C. for 1 hour with [3H]-cholesterol (2 μCi/ml) following by cell wash and a further incubation for 3 hours at 37° C. with or without non-treated HDL fractions (100 μg of protein/ml) or with non-treated LPDS fractions (at similar paraoxonase activity as in HDL). The cells were also incubated with HDL or LPDS fractions that were preincubated for 1 hour at 37° C. with the PON1 inhibitor 2-hydroxyquinoline (200 μM), or with HDL and LPDS that were enriched with evolved PON1 (50 paraoxonase U/ml). HDL or LPDS-mediated cholesterol efflux was then determined. Results are given as mean±S.D of three different experiments *p<0.01 vs. HDL, #p<0.01 vs. LPDS.
Patent Application
20070224657
