Josette Sainte-Marie

Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes.

We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experimental conditions (8 SH/LDL), the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.

The influence of coupling transferrin to liposomes or minibeads on its uptake and fate in leukemic L2C cells

Coupling transferrin to liposomes or minibeads did not affect its uptake by L2C lymphocytes via the Tf specific receptors. The uptake kinetics of Tf conjugated with particles about 50 nm in diameter was as rapid as in the case of native Tf, and the receptors were recycled with a similar turnover time (about 15 min). Contrary to the generally accepted scheme, we found some Tf degradation provoked by cellular uptake. The degradation represented about 10% of the amount of ligand taken up by the cells. It occurred when transferrin was coupled to liposomes, but not when coupled to minibeads.

Regulation of cholesterol biosynthesis at a stage after the 3-hydroxy-3-methylglutaryl-CoA reductase step, in normal and leukemic (L2C) guinea pig lymphocytes

Cholesterogenic activity in normal and leukemic guinea pig lymphocytes was measured by incorporation of labeled sodium acetate into cholesterol, after separation from other labeled metabolites. Our study is in agreement with the large difference previously found between the two kinds of cells at the 3-hydroxy-3-methylglutaryl-CoA reductase step, but it also shows that the difference is not as great as described earlier, when expressed in terms of the final product, cholesterol. This is mainly due to differences in the analytical methods. Our more detailed procedure showed a blockage of cholesterol synthesis in leukemic guinea pig lymphocytes (L2C cells) at the step of lathosterol (cholest-7-en-3 beta-ol) isomerization, and a higher plasma membrane permeability of these cells for sodium acetate, compared to normal cells. The lack of cholesterogenesis regulation by low density lipoproteins in L2C cells, previously reported after measuring 3-hydroxy-3-methylglutaryl-CoA reductase activity, was confirmed with regard to cholesterol itself, as well as the usual regulation of normal cells, which appeared to occur also at a post-hydroxymethylglutaryl-CoA step.

Characterization of low density lipoprotein receptors in freshly isolated leukemic guinea pig lymphocytes (L2C).

The present study shows that L2C leukemic guinea pig lymphocytes have 10 times as many low density lipoprotein (LDL) receptors per cell as normal lymphocytes. The affinity of these receptors is higher for guinea pig LDL than for human LDL. In contrast to normal cells, in which the degradation of the receptor-bound LDL is quite efficient, the leukemic cells only degraded a small fraction of these same receptor-bound LDL. Thus, the internalization index was nearly 4 times higher in the normal cells than in the leukemic cells. In L2C cells, cholesterol homeostasis derived 38% of its cholesterol input from receptor-mediated degradation of LDL and 62% from cholesterol synthesis, whereas in normal cells, these fractions were 97% and 3% respectively.

Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein

Low-density lipoproteins isolated between density 1.02 and 1.063 g/cm3 from normal fasting human plasma, show strong resonance Raman spectra due to the presence of beta-carotene. Three intense bands, at 1010, 1160 and 1530 cm-1, are assigned to the stretching vibrations of -C-CH3, = C-C = and -C = C- bonds, respectively, of beta-carotene. High-resolution spectra of the 1500-1600 cm-1 region reveal multiple features, suggesting the coexistence of several structural populations of beta-carotene. The modifications of lipoproteins with pH and temperature (30 degrees-42 degrees) change the resonance Raman spectra of beta-carotene. The specific binding of LDL at pH 7.0 by fibroblast cells is suppressed. Our experiments thus suggest that physical and chemical perturbations of plasma lipoproteins modify the lipid-protein interactions and thereby alter the configurational distribution of beta-carotene molecules within these particles.

Structural Abnormality in LDL From Diabetic Patients as Revealed by Resonance Raman Spectroscopy

Resonance Raman spectra of low-density lipoprotein (LDL) isolated from the plasma of diabetic patients (age range 13–77 yr, mean age 37 yr) and age- and sex-matched control subjects were recorded in the 1000- to 1600-cm−1 region as a function of temperature (0–50°C). Both nondiabetic and diabetic LDL yield spectra characterized by two major bands near 1160 and 1530 cm−1 due to the carotenoid component of lipoproteins. The relative intensity of 1530- and 1160-cm−1 bands, assigned to —C=C— and =C—C= stretchings, respectively, i.e., I1530-I1160 ratio, was plotted against temperature. For nondiabetic control subjects, the plots showed an inflection in the temperature range of 30–39°C, which corresponded to the thermal transition of LDL. This transition was abolished in the LDL of diabetic patients (P < 0.001), suggesting an altered lipid structure. The transition (30–39°C) was also abolished in the in vitro glycosylated nondiabetic LDL. Lipid analysis did not show any appreciable change between nondiabetic control subjects and diabetic patients. The change in the thermal transition properties of diabetic LDL has been attributed to the organizational change in the LDL protein.

Modifications of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fetal calf serum

The efficiency of supplying cholesterol by the LDL endocytic pathway of lymphoblastic T CEM cells was compared when incubated in the presence of either fetal calf serum (FCS) or lipoprotein-depleted fetal calf serum (LDFCS). In the presence of FCS, there were 8600 +/- 2000 LDL receptors/cell with a Kd of (2.2 +/- 0.8).10(-8) M and a receptor cycling time of about 7 min; about 90% of the internalized LDL was degraded. LDL degradation produced 98% of total cellular cholesterol and only 2% came from endogenous synthesis. The absence of LDL in the culture medium of lymphoblastic CEM cells deeply modified certain metabolic and structural characteristics of the cells. Their cholesterol content decreased; the total number of LDL receptors increased 6-fold, whereas their affinity for the ligand decreased by the same factor (Kd = (1.2 +/- 0.2).10(-7) M); the receptor cycling time increased 3-fold. Finally, LDL degraded by cholesterol-depleted CEM cells amounted to about 40% of that degraded by untreated CEM cells.

Raman studies of structural rearrangements induced in human plasma lipoprotein carotenoids by malondialdehyde.

Raman and resonance Raman spectra of plasma lipoproteins ± malondialdehyde were studied at concentrations which block the normal receptor-mediated uptake by cells. The strong resonance Raman bands at about 1010, 1162 and 1530 cm−1, due to the presence of carotenoids in the lipoproteins, are envisaged as structural probes. High resolution resonance Raman spectra of the 1500–1600 cm−1 region reveal multiple features suggesting the coexistence of several structural populations of β-carotene whose precise assignment is complex. When plasma lipoproteins are reacted with malondialdehyde, a complex change occurs in the resonance Raman banding of β-carotene in the 1500–1600 cm−1 region. Malonaldehyde (MDA) also modifies the acoustical region (70–200 cm−1 of low density lipoprotein (LDL) lipids. We suggest that malondialdehyde association with plasma lipoproteins alters the lipid structure via apoprotein or apoprotein/lipid associations.

Kinetics of phospholipid transfer between liposomes (neutral or negatively charged) and high-density lipoproteins: a spin-label study of early events

The kinetics of spin-labeled phosphatidylcholine transfer between vesicles and HDL particles exhibited a two-phase process, as seen by ESR spectroscopy. The results were analyzed by considering several possible steps in the overall transfer, whose aspects were also studied: (i) micellar complex formation after HDL apolipoprotein-vesicle mixture, (ii) the rate of PC transfer from the micellar complex to HDL, (iii) the rate of the reverse reaction between overloaded HDL particles and other particles such as HDLs, LDLs, and lipid vesicles. The results agree most convincingly with a mechanism in which the diffusion of phospholipids into the HDL-endogenous lipids is the limiting step, occurring as a two-step process. In addition, we observed a negative charge effect on the lipid transfer rates and yields.

Comparison of the internalization efficiency of LDL and transferrin receptors on L2C guinea pig lymphocytes

We demonstrate that L2C lymphocytes have about 10‐times more receptors for transferrin (TO than healthy lymphocytes, as has been shown in the case of LDL receptors. The dissociation constant is the same in the two cell types (about 4 × 10−7 M). In contrast to LDL, Tf enters L2C lymphocytes with very rapid kinetics. It is shown by cross‐reaction that each receptor is internalized independently of the other.