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M Mersel

Publications and source records attributed to M Mersel.

At least 37 records · Page 2Linked to original sources
PubMed1989

Toxic effects of 7 beta-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures.

The toxic effects of 7 beta-hydroxycholesterol (7 beta-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 beta-OHC at a concentration of 400 microM over a period of 72 hours. In contrast, proliferative cultures of liver epithelial cell lines and liver fibroblast lines were killed by 50 microM 7 beta-OHC within the first 24 hours. Established liver epithelial cells (hyperploid) were more sensitive to 7 beta-OHC than the same line at early passages (diploid). When hepatocytes and liver epithelial cells were co-cultured and treated with 100 microM 7 beta-OHC, only epithelial cells were lysed. A concentration of 50 microM 7 beta-OHC was toxic to co-cultures of liver epithelial cell and fibroblasts together. In a serum-free medium, the cytotoxic concentration of 7 beta-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 microM and 50 microM 7 beta-OHC, respectively. Finally, cholesterol concentrations four-fold that of 7 beta-OHC antagonized the lethal effects of 7 beta-OHC in the serum-free medium.

Albumins↗
PubMed1988

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines. Effect of dibutyryl cyclic AMP treatment.

Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.

Animals↗
PubMed1988

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

7 beta-OH cholesterol in a cholesterol rich growth medium (5-10 micrograms/ml) extended the lag period and slowed down the growth rate of Mycoplasma capricolum cells. In a cholesterol poor medium (0.5 micrograms/ml) inadequate to support growth, 7 beta-OH cholesterol exerts a synergistic effect on growth. The 7 beta-OH cholesterol was incorporated unchanged from the growth medium and could be recovered exclusively in the membrane fraction. The incorporation of the 7 beta-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 7 beta-OH cholesterol was exchanged.

Cardiolipins↗
PubMed1988

Studies on oxygen and volume restriction in cultured cardiac cell: possible rearrangement of sarcolemmal lipid moieties during anoxia and ischemia-like states.

Cultured heart cells have been shown useful for investigating states of oxygen and volume restrictions, simulating anoxia and ischemia-like states at cellular levels. The sarcolemma has been implicated as one of the early sites of ischemic damage; therefore, lactoperoxidase catalyzed radioiodination was used to study accessibility of the sarcolemmal lipid moieties to this enzymatic probe, reflecting their exposure to the extracellular environment, hence the biophysical state of the sarcolemma. These studies have shown that within one hour of 'ischemic' injuries: (1) The degree of labelling in the total phospholipid fraction is considerably increased; and (2) Profound changes in the relative extent of labelling of different phospholipid classes were observed. The PE/PC labelling ratio increases dramatically with the progress of ischemia-like state. We suggest that early during ischemic injury, reorganization of the cell surface phospholipids occurs and discuss possible relations to the energy charge of the cell.

Animals↗
PubMed1988

The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.

One of the neuronal growth-associated proteins, GAP-43 (molecular mass, approximately 43 kDa; pI 4.3), is abundant in growth-cone membranes and corresponds to a major protein kinase C substrate, the 46-kDa phosphoprotein (pp46), of a growth-cone-enriched subcellular fraction. This protein has the following additional designations (depending on context): B-50 (phospholipid metabolism), F1 (synaptic plasticity), and p57 (calmodulin binding). We show that a protein with the same molecular mass and isoelectric point as GAP-43, which interacts with anti-GAP-43 antibodies on immunoblots, is present in the plasma membranes of cultured neonatal rat cortical astrocytes. Double-immunofluorescence labeling of cells with a serum against glial fibrillary acidic protein and anti-GAP-43 antibody was observed. Furthermore, astrocytic protein was phosphorylated in vitro by protein kinase C and comigrated in two-dimensional PAGE with GAP-43. The data indicate that GAP-43, heretofore believed to be neuron-specific, is present in at least one class of glial cells.

Animals↗
PubMed1987

Isolation of plasma membranes from neurons grown in primary culture.

Plasma membranes from chick embryo neuronal primary cultures were isolated after subjecting 5-day-old cells, previously surface labeled with either lactoperoxidase-catalyzed radioiodination or galactose oxidase/NaB3H4, to a freeze-thaw cycle. The cellular material adhering to the culture substratum was washed, and the "wash" fractions were pooled and centrifuged at 37,000g. The resulting pellet was resuspended in 3 ml of buffer, layered on 33 ml of 33% sucrose, and centrifuged at 105,000g. Radioactivity was recovered at the top of the gradient. Sedimentation of these fractions and biochemical studies revealed that the pellet was 20- and 12-fold enriched in (Na+,K+)-adenosinetriphosphatase and 5'-nucleotidase, respectively. The preparation was devoid of inner mitochondrial (succinate dehydrogenase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), and Golgi (UDP galactose:N-acetylglucosamine galactosyltransferase) enzymatic markers. Ultrastructural studies showed that the membrane preparation was homogeneous and lacked mitochondria endoplasmic reticulum and lysosomes. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of 11 protein components with molecular masses ranging from 120 to 300 kDa. This method for the isolation of plasma membranes probably depends on the capacity of the cellular material to adhere to the culture substratum and to entrap intracellular organelles during the freeze-thaw cycle. The membrane preparation seems suitable for studying the function of high-molecular-weight protein components of neuronal plasma membranes.

5'-Nucleotidase↗
PubMed1987

Topological distribution of aminophospholipid 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.

Animals↗
PubMed1987

Ethanolamine base exchange in astrocyte primary cultures: localization and developmental studies.

The enzymatic activities of ethanolamine base exchange (EBEE) and CDP-ethanolamine: 1,2-diacylglycerol ethanolamine phosphotransferase (EPT) were investigated during the growth of rat astrocyte primary cultures. From the 16th day, cells ceased to divide (2.0 X 10(6) cells per culture dish); the total phospholipid (PL) content increased 1.5 fold between the 16th and 24th day (0.20 to 0.30 mumol per mg protein) but the amount of ethanolamine phospholipid (28% of PL content) remained constant. Whereas the specific activity (pmol/min X mg protein) of EPT reached a plateau at 16 days in culture and remained constant (400) thereafter, that of EBEE increased up to the 19th day (190) and decreased gradually to a basal level (75) at the 24th day. EBEE activity was not detected in plasma membranes isolated from 16, 19 and 24 days astrocyte cultures. Sub-cellular fractionation and determination of EBEE specific activities showed that the 104 X 10(3) g fraction (P4) was 4.8 and 8.8 fold enriched at the 16th day and 24th day respectively as compared to the whole cell homogenate (50 and 75). The 7 X 10(3) g (P2) and 17 X 10(3) g (P3) fractions were 8.4 and 7.0 fold enriched respectively at the 19 day in culture. The percentages of the enzymatic activity in the different subcellular fractions were 30, 57.2 and 25.7 for P2 and 39.2, 2.6 and 39.8 for P4 at 16, 19 and 24 days in culture respectively. The activity remained constant in P3 (23%) and was negligible in P1 (6%). Ultrastructural studies revealed that P2 and P3 were enriched in mitochondria while P4 contained essentially microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Phospholipid fatty acyl group composition in mitochondria, microsomes and plasma membranes isolated from rat astrocyte primary cultures: developmental studies.

The fatty acid composition of choline, ethanolamine and serine phospholipids (PC, PE and PS) in heavy mitochondria, light mitochondria, microsomes and plasma membranes isolated from 16-, 19- and 24-day-old cultured rat astrocytes was investigated; within this period cultures reached confluency and ceased to divide. Plasma membranes revealed a stable distribution of the major fatty acids (FA) present in PC, PE and PS. The FA pattern of PC was similar to that of PE and PS. Analysis of the FA composition of PC, PE and PS found in intracellular membranes indicates that each major FA displayed a typical variation depending on the organelle and the phospholipid studied. Schematically for both phospholipids, PC and PE, the palmitic acid content increased in most subcellular fractions between 16 and 24 days; this variation was compensated by the decrease of stearic acid. Arachidonic acid level was very low at 16 days and increased markedly up to 24 days; thus a decrease of the saturated to unsaturated FA ratio (delta:0/delta:1, 2, 4) occurred. In contrast PS, which was poor in arachidonic acid between 16 and 24 days, gave rise to delta:0/delta:1, 2, 4. These results suggest (1) a reorganisation of the phospholipid hydrophobic core in mitochondria and endoplasmic reticulum when density-dependent inhibition phenomena occurs, and (2) a 'buffering' mechanism which protects the plasma membrane from marked changes of its phospholipid FA composition.

Animals↗
PubMed1986

The nature of DT-diaphorase (EC 1.6.99.2) activity in plasma membrane of astrocytes in primary cultures.

This is the confirmation of an earlier indication (Mersel, M., Malviya, A.N., Hindelang, C. and Mandel, P. (1984) Biochim. Biophys. Acta 778, 144-154) that the plasma membrane of astrocytes in primary cultures is endowed with DT-diaphorase (EC 1.6.99.2) activity. It is observed that the NADPH-2,6-dichloroindophenol diaphorase activity found in the isolated plasma membrane is not inhibited by dicoumarol. DT-diaphorase-type activity is also observed on the cell surface employing dichloroindophenol as external electron acceptor and it is found to be a dicoumarol-sensitive NADH dehydrogenase.

Animals↗
PubMed1985

[Isolation and characterization of a new cell line of spontaneously transformed astrocytes of the rat brain].

A new cell line of transformed astrocytes was obtained from primary new born rat brain cultures. rat brain cultures. These transformed cell line possess a normal karyotype, a doubling time of 17-24 hrs. and astrocyte specific protein marquers: glial fibrillar acidic protein (GFA) and glutamine synthetase. The ganglioside pattern is more differentiated than that of other known astrocyte lines.

Animals↗
PubMed1984

Plasma membrane isolated from astrocytes in primary cultures. Its acceptor oxidoreductase properties.

Rat astrocytes in primary cultures were employed to isolate the plasma membrane. The method for the isolation of plasma membrane was based on the capacity of the cytoskeleton to adhere to the substratum entrapping intracellular organelles during freezing-thawing cycle performed on the cell. By washing the 'surface adherent framework', the untrapped plasma membrane were recovered and density equilibrium centrifugation resulted in the isolated membrane. The isolated plasma membrane was characterized on the basis of a variety of marker enzymes positive to the plasma membrane such as (Na+ + K+)-ATPase or 5'-nucleotidase as well as the lack of conventional markers of other endomembranes. Ultrastructurally the membranes, as isolated here, were mainly vesicular in nature. The isolated plasma membrane was devoid of the dehydrogenase responsible for NADH-cytochrome c reductase activity. However, NADH-ferricyanide reductase activity and the dehydrogenase system catalyzing the transfer of reducing equivalents from NADH or NADPH to dichloroindophenol seems plasma membrane redox system. The identical specific activity employing dichloroindophenol as an electron acceptor with NADH or NADPH as donor indicate a DT-diaphorase (EC 1.6.99.2) like activity in the astrocytes plasma membrane.

5'-Nucleotidase↗
PubMed1984

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.

Ammonia↗
PubMed1984

[Differential action of 7 beta-hydroxycholesterol on myocardial cells and fibroblasts. Obtaining primary cultures of pure myocardial cells].

Newborn rat myocardial cells, grown in primary cultures, beat synchronously. Addition of 7 beta-hydroxycholesterol, at a 2.5 microM concentration, impairs this synchrony and may even stop any contraction. The associated fibroblasts no longer adhere to the support, and can be washed away by fresh culture medium. This restores the synchronous beatings of the myocardial cells, the viability of which is then even improved while they grow in the absence of fibroblasts.

Animals↗
PubMed1983

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid.

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium welchii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.

Animals↗
PubMed1983

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 TDC3, 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.

Animals↗