Jacques Polonovski

In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein, and its delivery to cultured human fibroblasts

Low density lipoprotein (LDL) doped with the anticancer mixture of hematoporphyrin derivatives Photofrin II (P2) competes with native LDL for binding to fibroblast receptors, despite a slight increase in the negative net charge related to the presence of acidic residues of porphyrins. P2 delivery to fibroblasts can be achieved by LDL, HDL3 or albumin doped with P2 (LDL‐P2, HDL‐P2 or A‐P2, respectively). P2 delivery to cells assessed by fluorescence measurement, is much more efficient, at low protein concentrations (10–20 ) by LDL‐P2 than by HDL‐P2 or A‐P2. Moreover, P2 delivery to cells by LDL‐P2 as a function of protein concentration is a saturable process, whereas P2 delivery by HDL‐P2 or A‐P2 is a linear process. Finally, reduction of the LDL‐receptor number by preincubation of fibroblasts in medium supplemented with lipoproteins results in a decrease of P2 delivery by LDL‐P2. These results suggest a special role of the LDL‐receptor pathway in P2 delivery to cells and could be of interest in cancer phototherapy by porphyrins.

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Abstract Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-14C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the in situ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in vitro by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.

Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo

We describe an ELISA technique able to recognize malondialdehyde-modified low-density lipoproteins (LDL). For this purpose we produced antibodies to malondialdehyde-LDL, specific for the malondialdehyde modification of LDL; these antibodies recognized essentially malondialdehyde-LDL. Coating ELISA plates with the antibodies to malondialdehyde-LDL and using peroxidase-labelled antibodies to LDL, which reveal only apolipoprotein B, we obtained an accurate method of detecting malondialdehyde-modified apolipoprotein B. Preliminary studies demonstrated that this method allows the detection of lipoproteins containing malondialdehyde-modified apolipoprotein B in the serum of patients with cardiovascular diseases.

Rapid analysis of cellular lipids without extraction

A method is described where cell suspension obtained by scraping of monolayers was directly applied on silica gel plates. Extraction and separation of different lipid classes were simultaneously obtained during chromatography. In the range of validity of the method (no more than 80 micrograms of cellular protein tested for neutral lipids and 30 micrograms for phospholipids), this technique allows rapid lipid analysis of small samples of cultured cells, bypassing all the time- and solvent-consuming extraction and evaporation steps. The method appears to be also suitable for measurement of enzymes of lipid metabolism such as acyl coenzyme A-cholesterol-acyltransferase.

Acylation of endogenous phospholipids and added lysoderivatives by rat liver plasma membranes

Phospholipid acyltransferase activities of plasma membranes have been investigated with various acyl-CoA thioesters (palmitoyl, stearoyl, oleoyl, linoleoyl and arachidonoyl) with and without added lysoderivatives. Different patterns of incorporation were observed for each acyl-CoA into endogenous phosphatidylcholine and phosphatidylethanolamine. The turnover rates calculated with tracer amounts of 10 microM acyl-CoA thioesters were five times faster for the polyunsaturated than for the saturated acyl moieties of phosphatidylethanolamine and phosphatidylcholine. Arachidonoyl-CoA was the best acyl donor at low concentrations and the maximal turnover rate was observed at about 25 microM. No saturation appeared at up to 100 microM linoleoyl-CoA. Linoleoyl-CoA transacylase acylated the lyso-compounds in the following order: lysophosphatidylcholine greater than lysophosphatidylserine and lysophosphatidylinositol, while lysophosphatidylethanolamine inhibited linoleate incorporation into the phosphatidylethanolamine itself. Linoleoyl-CoA transacylation was not affected by the fatty acyl moiety at the 1-position of the lysophosphatidylcholine. The results support the view that the plasma membrane acyltransferase activity might contribute to the formation of bile phosphatidylcholines.

Phospholipase activities in subcellular fractions of human platelets.

A homogenate of human platelets was fractionated by zonal ultracentrifugation into membranes, various granules and mitochondria. The membrane fraction was composed of two populations. The first, which represented 75% of the proteins, was rich in plasma membranes; the second, which represented the remaining 25%, was rich in microsomal membranes. Lysophospholipase was essentially localised in the cytosol. Phospholipase A1 which was only weakly bound to membranes, was mostly found in the soluble fraction (75%); the remainder was located in the plasma membranes and the mitochondria. Two-thirds of the phospholipase A2 was found in the particulate fractions.

Elastase-type activity associated with high density lipoproteins in human serum

Abstract Elastase-type activities were found associated with lipoprotein fractions in human serum. A metallo-protease hydrolysing Suc(Ala) 3 PNA and soluble elastin peptides was isolated from apoHDL by Sepharose-elastin chromatography. Polyacrylamide gel electrophoresis and immunoelectrophoresis indicated that this elastase-type activity was associated with apoA 1 .

Influence of chain length and degree of unsaturation on plasma free fatty acid uptake by the perfused rat liver

Abstract 1. 1. The fractional uptake of free fatty acids by perfused liver is inversely related to the chain length and directly related to the number of double bonds in the molecule. 2. 2. The affinity of the liver for long chain free fatty acids is myristic > palmitoleic > linoleic > oleic ≅ palmitic > stearic. Partial in vitro extraction by charcoal of fatty acids bound to albumin shows similar behaviour supporting the view that the first step in the uptake of fatty acids by the cell is a non-selective, physical adsorption. Therefore, it is suggested that the rate of fractional uptake of the individual free fatty acids depends less on the selectivity of absorption to the cellular membrane than on the relative affinity of the individual fatty acids for the binding sites on the albumin molecule. 3. 3. The uptake of the total exchangeable pool of plasma free fatty acids by the liver can be measured rather accurately by using oleic or palmitic acid as tracer. 4. 4. The difference in the uptake of individual fatty acids could explain the finding that the proportion of stearic acid in the plasma is greater than in adipose tissue. At the same time, it can explain the relative variations (increase of stearic acid and decrease of oleic acid) observed when there is a fall in the plasma free fatty acids concentration.

Etude des phospholipases A des membranes plasmiques de foie de rat

Summary The plasma membranes phospholipase A2 studied in situ shows very little sensitivity for pH variations; the optimal concentration for Ca++ is 5 mM; the enzymatic kinetics are of the Michaelis type. An inactive state of the phospholipase A2 exists: when rats are injected with heparin, their plasma membranes contain a very low phospholipase A2 activity. These membranes recover a high A2 activity if they are incubated in the presence of a rat platelets lysate. Treatment of membranes by NaCl 1 M displaces phospholipase A1 and phospholipase A2 but for the latter only when being in active state. Treatment of animals, conditions of preparation and of incubation of membranes influence the respective amounts of phospholipases A1 and A2 present in these membranes and are discussed in this paper. The localisation of these enzymatic proteins inside the membrane according to the membranous model proposed by Singer et al. [18] is also discussed.

Trifluoperazine increases fatty acid turnover in phospholipids in cultured human fibroblasts

A 24-hr pretreatment of cultured human fibroblasts with trifluoperazine induced a marked increase in incorporation of saturated (stearic, palmitic) and unsaturated (oleic, arachidonic) fatty acids into phospholipids (1.5- to 2-fold for 5.10−5 M trifluoperazine). Concomitantly, incorporation into cholesteryl esters was strongly inhibited (20% of control for 5.10−5 M trifluoperazine). The drug did not change the phospholipid composition of treated cells. The effect of trifluoperazine on oleic acid incorporation into phospholipids was time-dependent and reached a maximum after a six-hr preincubation with the drug. Trifluoperazine also induced an increase in the rate of chase of oleic acid from the different phospholipid classes. In vitro preincubation of cell-free extracts with trifluoperazine resulted in activation of phospholipid acyltransferases, whereas cholesterol acyltransferase activity was decreased. The rapid effect of trifluoperazine together with its effect on a cell-free system suggests a direct action of this amphiphilic drug on the acyltransferase activities, probably by modification of the structural organization of cellular membranes.