M. Mersel

Rat liver chromatin phospholipids

To shed light on the question whether the phospholipids present in chromatin are native or are due to contamination from nuclear membranes, we labeled the phospholipids of isolated nuclei and determined the amount of phospholipids (PL) and PL fatty acid composition in nuclei and chromatin. The hepatocyte nuclei were isolated and radioiodinated by the lactoperoxidase method under saturating and nonsaturating conditions, and the radioactivity associated with chromatin extracted from these nuclei was monitored. Whereas 97% the label was recovered in the nuclear membranes, only 0.08–0.6% was found in chromatin. The PL present in chromatin were relative to the amounts present in the entire nuclei and calculated as percentage of total, phosphatidylethanolamine (10%), phosphatidylserine (22%), phosphatidylinositol (19%) phosphatidylcholine (14%), and sphingomyelin (35%). In sphingomyelin of chromatin-associated PL an enrichment in polyunsaturated fatty acids was seen. The data indicated that the PL found in isolated chromatin do not seem to be due to contamination from the nuclear membrane.

Effect of Oxysterol Treatment on Cholesterol Biosynthesis and Reactive Astrocyte Proliferation in Injured Rat Brain Cortex

Abstract: We have reported previously that oxysterols inhibit astrogliosis and intracranial glioblastoma growth. To elucidate the mechanism of action of these molecules in vivo, we have investigated their effect on the cholesterol biosynthesis in the injured brain. In a bilateral lesion model, injection of liposomes containing 7β‐hydroxycholesterol decreased [3H]acetate incorporation into neutral lipids and cholesterol by 30% and 40%, respectively. Structural analogues were tested using a unilateral lesion model. The injury did not significantly affect cholesterogenesis; injection of 7β‐hydroxycholesterol or 7β‐hydroxycholesteryl‐3‐oleate reduced acetate incorporation into cholesterol by 47% and 43%, respectively. Both 7‐ketocholesteryl‐3‐oleate and 7α‐hydroxycholesteryl‐3‐oleate inhibited cholesterogenesis by 32%. As cholesterol and by‐products of the cholesterol pathway play a key role in cell division, we have assessed the effect of oxysterols on reactive astrocyte proliferation. The incorporation of bromodeoxyuridine showed that up to 46% of astrocytes were proliferating 24 h after the injury. Injection of 12 nmol of 7β‐hydroxycholesterol or 7β‐hydroxycholesteryl‐3‐oleate reduced the labelling index to 26%, whereas the labelling index in the 7‐ketocholesteryl‐3‐oleate‐treated cortex was 37%. These findings demonstrate that oxysterols are potent inhibitors of the endogenous cholesterol biosynthesis in brain and show a correlation between cholesterogenesis and reactive astrocyte proliferation.

7β-Hydroxycholesterol and 7β-hydroxycholesteryl-3-esters reduce the extent of reactive gliosis caused by an electrolytic lesion in rat brain

Abstract Electrolytic lesions performed in brain cortex of six-day-old or adult rats resulted in the appearance of many reactive astrocytes around the injury site after a postoperative delay of eight days. They were revealed by immunohistochemistry using antibodies against glial fibrillary acidic protein. Injection of tritiated thymidine 24 h prior to autopsy indicated that, in neonates, 50% of the reactive astrocytes were proliferating. Infusion of 2 μl of liposome suspension made of phosphatidylcholine and a monosialoganglioside, in the injury site, immediately after the electrolytic lesion did not modify the extent of the reactive gliosis. Liposomes containing 3 nmol of either 7β-hydroxycholesterol, 7β-hydroxycholesteryl-3-stearate or 7β-hydroxycholesteryl-3-oleate reduced by about 50% the intensity of the reactive gliosis in the frontal cortex of six-day-old rats and by 40% the number of dividing astrocytes. In the adult rat cortex the intensity of the glial reaction was also decreased by 30% by 15 nmol 7β-hydroxycholesteryl-3-oleate. Further investigations demonstrated that it is the 7β-hydroxy function which is needed for the biological activity of these oxysterols. These findings, which demonstrate anti-proliferative and anti-inflammatory properties of 7β-hydroxycholesterol on astrocytes, facilitate the future investigation of the influence of reactive gliosis on functional recovery following brain injury. This anti-proliferative property could also be used in other kinds of pathologies involving glial cell proliferation, such as glioblastomas.

Long-term effects of brain trypsinization before cell seeding on cell morphology and surface composition

The relation between the pattern of proteins localized in the surface of astroglial cells and cell differentiation was investigated in primary cultures derived from neonatal rat brains, dissociated either mechanically (MDC) or by 3 (TDC3) and 30 minutes (TDC30) trypsinization. Morphological and ultrastructural studies revealed a bed layer composed of flat, polygonal young and differentiated astrocytes in all types of cultures and a surface layer composed of small, ovoide undifferentiated cells which were more numerous in TDC30 than in TDC3 and MDC. The enrichment in undifferentiated cells, induced by prolonged brain trypsinization prior cell seeding, was observed during two weeks in culture: latter, by day 20, the cell population in all cultures was that of differentiated astrocytes. The presence of structural and enzymatic cell markers indicated that the cell population in MDC and TDC3 as well as in TDC30, including the small cells, was of astroglial origin. Concomitant with the morphological changes, cells in TDC30 were less accessible to surface labeling than those composing MDC. Subsequent electrophoresis of the labeled surface proteins demonstrated that a 140–130 K complex was the most “sensible” to brain trypsinization and that their accessibility to the surface probing was maximal during the differentiation of astrocytes in MDC or of small cells in TDC30. By day 20, these components were not significantly labeled in both, MDC, and TDC30, cultures. The use of two types of astrocytes primary culture which were different in the ratio of differentiated to undifferentiated cells and their surface labeling at different growth stages showed a variation in the composition of surface proteins during the cell maturation. The increased accessibility of some surface proteins to external probing when the cells developed to differentiated astrocytes might suggest their involvement in cell differentiation.

Topological distribution of choline phospholipid fatty acids in trout intestinal brush-border membrane

The transbilayer distribution of aminophospholipids in trout intestinal brush-border membrane has been investigated using trinitrobenzene sulfonic acid (TNBS). In the middle intestine, phosphatidylethanolamine (PE) is symmetrically distributed between the two leaflets while 68% of the phosphatidylserine (PS) are located in the inner membrane leaflet. In the posterior intestine, 64% of the PE and 69% of the PS are located in the inner membrane leaflet. When asymmetrically distributed, the inner species of PE and PS have a higher content of 22:6(n-3) than the outer ones. This asymmetric distribution of docosahexaenoic acid in trout intestinal brush-border membrane might be related to the rod-like shape of the microvillus membrane and to its metabolism to hydroxylated derivatives.

Toxic effects of 70-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures.

The toxic effects of 7,8-hydroxycholesterol (7β-OHC) on cultures and co-cultures of rat hepatocytes, rat liver epithelial cell lines, and rat liver fibroblast lines were investigated. Hepatocytes in primary culture or co-cultured with proliferative epithelial cells, were not affected by the presence of 7,8-OHC at a concentration of 400µM over a period of 72 hours. In contrast, Proliferative cultures of liver epithelial cell lines and liver fibroblast lines were killed by 50 µM 7β-OHC within the first 24 hours. Established liver epithelial cells (hyperploid) were more sensitive to 7,8-OHC than the same line at early passages (diploid). When hepatocytes and liver epithelial cells were co-cultured and treated with 100 µM 7β-OHC only epithelial cells were lysed. A concentration of 50 µM 7β-OHC was toxic to co-cultures of liver epithelial cell and fibroblasts together. In a serum free medium, the cytotoxic concentration of 7β-OHC was lower than that in the serum-supplemented medium. Thus, liver epithelial cells cultured alone or co-cultured with hepatocytes were killed at 12.5 µM and 50 µM 7β-OHC, respectively. Finally, cholesterol concentrations four fold that of 7β-OHC antagonized the lethal effects of 7β-OHC in the serum free medium.

Effect of 7β-hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum

7β‐OH cholesterol in a cholesterol rich growth medium (5–10 μg/ml) extended the lag period and slowed down the growth rate of Mycoplasma capricolum cells. In a cholesterol poor medium (0.5 μg/ml) inadequate to support growth, 7β‐OH cholesterol exerts a synergistic effect on growth. The 7β‐OH cholesterol was incorporated unchanged from the growth medium and could be recovered exclusively in the membrane fraction. The incorporation of the 7β‐OH cholesterol has no effect on the total phospholipid content but the DPG to PG ratio was markedly decreased. Exchange studies with lipid vesicles revealed that whereas most of the cholesterol underwent exchange, only about 20% of the 7β‐OH cholesterol was exchanged.

Inhibition of p42/p44 mitogen-activated protein kinase by oxysterols in rat astrocyte primary cultures and C6 glioma cell lines

We previously demonstrated that oxysterols inhibit the growth of experimental glioblastoma induced in the rat brain cortex. Mechanism of action of these compounds remains obscure. In this study, we investigated the effect of 7β‐hydroxycholesterol (7β‐OHCH) and 7ketocholesterol (7k‐CH) on the growth and MAP kinase activity in three in vitro biological models: rat astrocyte primary cultures, primary cultures treated by dibutyryl‐cAMP (reactive cells), and the C6 glioma cell line. The oxysterols are not lethal to primary astrocytes, even if MAP kinase activity is decreased, particularly when cells were treated with 7k‐CH. Both oxysterols are toxic to reactive astrocytes, and as compared with untreated primary cultures, they amplified the MAP kinase activity decrease. However, the mechanism of action of oxysterols on reactive astrocytes seems not to be linked to the MAP kinase pathway. In highly proliferating C6 cell lines, only 7β‐OHCH has an antiproliferative effect and is cytotoxic. The inhibition of MAP kinase activity is a function of 7β‐OHCH concentration. PD098059, a MAP kinase pathway inhibitor, has only a time‐limited antiproliferative effect on C6 cell growth. We conclude that in C6 cells, the MAP kinase activity decrease is correlated with the toxic effect of 7β‐OHCH and occurs at first stages of 7β‐OHCH action. J. Neurosci. Res. 53:38–50, 1998.

Fast-heating mass spectrometry of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, and sphingomyelin

A fast-heating probe and chemical ionization have been used to obtain mass spectra of the synthetic glycerophospholipids 1,2-diacyl-sn-glycero-3-phosphocholine, 1,2-diacyl-sn-glycero-3-phosphoethanolamine, and 1-monoacyl-2-lyso-sn-glycero-3-phosphocholine. The phospholipids investigated gave quasimolecular peaks and fragment ions, with preferential cleavage of the C-O bond (beta position to the phosphorus atom) and loss of phosphoethanolamine or phosphocholine. This technique makes possible the analysis of mixtures of intact phosphatidylethanolamine, phosphatidylcholine, 2-lysophosphatidylcholine, and sphingomyelin isolated from natural sources such as egg yolk or brain. Only minor and inexpensive modifications of a standard mass spectrometer are required.

Kinetic characterisation and solubilisation of γ-hydroxybutyrate receptors from rat brain

Abstract The solubilisation of the γ-hydroxybutyrate (GHB) receptors from rat brain membranes was undertaken as the first step for their molecular characterisation and purification. Treatment of crude brain membranes with high concentrations of NaCl and Triton X-100 resulted in solubilisation of proteins which retain specific GHB binding activity. Ionic detergents do not solubilise and/or inactivate the receptors. Measurements of kinetic parameters of GHB binding showed that the solubilised receptor, in the presence of detergent, exhibited a reduction of affinity for GHB and its endogenous brain analogue trans-4-hydroxycrotonate (T-HCA). The membrane protein extract, submitted to chromatography by gel filtration, showed a single peak of protein with [ 3 H]GHB binding activity. Association and dissociation constants of GHB for its membrane binding site were in accordance with the K d determined by the Scatchard method.