Expression of growth-regulated genes in normal and SV40 transformed hamster fibroblasts.

Transformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth-regulated genes in SV40-transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1-specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk-ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk-ts13/1%), was also included in these studies. The low serum adapted SV40-transformed cells and the spontaneous tk-ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

Metabolism of cholesterol in normal hamster fibroblasts and those transformed by the SV 40 virus. Effect of low density lipoproteins

The amount of cholesterol and the percentage of esterified cholesterol were increased in transformed cells. The cholesterol synthesis from [14C] sodium acetate was reduced and cholesteryl oleate uptake increased by 3 fold in transformed cells. The activity of acyl coenzyme A-cholesterol-acyltransferase, measured in situ was also increased in transformed cells. Studies with 125I-LDL pointed out an increase of binding, and especially of internalization of LDL by transformed cells. Finally, long term culture in a lipoprotein-deficient medium showed that transformed cells exhibited a higher ability (tested by growth rate and cholesterol synthesis) to adapt themselves to lipid depletion.     

Phospholipid composition and turnover in normal and SV40 transformed fibroblasts. Effect of cell density]

The phospholipid composition and turnover in normal and in SV 40 transformed hamster fibroblasts were studied. The amount of phospholipid phosphorus relative to protein is lower in transformed hamster fibroblasts than in normal fibroblasts. This amount decreases with increasing cell density until stationary growth is reached. The decrease is largest for the normal fibroblasts. In transformed cells, less sphingomyelin and more diphosphatidyl glycerol are found than in normal cells. The turnover of 32P in sphingomyelin is slower in transformed cells than in normal cells ; the contrary is observed with diphosphatidyl glycerol. On the other hand, in transformed cells, phosphatidyl ethanolamine has a faster turnover than phosphatidyl choline, whereas the contrary is observed in normal cells. Finally, the change to stationary growth slows down the turnover of 32P of all phospholipids, this decrease being more important in transformed cells.     

Fatty acid composition in the classification of Saccharopolyspora hirsuta

The long-chain fatty acids of 4 Saccharopolyspora hirsuta strains were examined as their methyl and picolinyl esters using gas liquid chromatography (GLC) and GLC-mass spectrometry (GLC-MS). All the strains had similar fatty acid profiles composed mainly of isoanteiso-, and 10-methyl-branched components. Three new substituted 10-methyl-branched fatty acids were also detected and the major component identified as 10,15-dimethylhexadecanoic acid. The implications of these data for Saccharopolyspora systematics are discussed.     

Cadmium effects on lipid metabolism of rape (Brassica napus L.)

Treatment of rape seedlings with increasing CdCl₂ concentrations in the culture medium resulted in a cadmium accumulation within plant tissues, which increased with external metal dose; such accumulation was more important in roots than in leaves. Biomass production was severely inhibited, even at low cadmium concentration. In leaves, quantities of chloroplastic lipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfolipids (SL) and phosphatidylglycerol (PG) decreased sharply under metallic treatment. However, contents of extrachloroplastic lipids, mainly phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased significantly. In contrast to leaves, contents of root phospholipids decreased. Likewise, levels of tri-unsaturated fatty acids: linolenic (C18:3) and hexadecatrienoïc (C16:3) dropped in leaves of treated seedlings as compared to those of controls, suggesting that heavy metals induced an alteration in the fatty acid desaturation process or a stimulation of their peroxidation. Also, trans palmitoleic acid (C16:1-trans) level in PG decreased considerably. In roots, there was a slight decrease in C18:3 level, with a concomitant increase in the C18:2 percentage. Radioactive labelling of leaf lipids with (1-¹⁴C) acetate allowed to show that fatty acid biosynthesis was noticeably altered at the highest cadmium dose used (50 μM). Biosynthesis of tri-unsaturated fatty acids was also inhibited which may explain the decline in non-labelled lipid contents. Results showed that metallic ion seems to affect selectively chloroplastic membranes due to an inhibition of polyunsaturated fatty acid biosynthesis. Moreover, a lipid peroxidation occurred in our case because of the spectacular increase of malondialdehyde (MDA) content observed in cadmium treated leaves. To cite this article: N. Ben Youssef et al., C. R. Biologies 328 (2005).     

A difference in the breakdown of phosphatidylinositol in normal and SV40 transformed mouse fibroblasts

Pulse chase experiments have shown that four reactions of phospholipid metabolism depend on cell population density in normal cells. Three of these reactions are influenced by population density also in tumor cells. Only the breakdown of phosphatidylinositol is not affected.     

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.     

Fatty acid and carotenoid composition ofRhodotorula strains

Lipid content and composition of fatty acids with 6–25 carbon atoms were studied on strains of the 13 pink or red yeast species belonging to the genus Rhodotorula. The total amount of lipid represented an average of 13% of the dry weight. The neutral and polar lipid fractions were analyzed separately. For all the strains studied, the major fatty acids in both fractions were oleic, linoleic and palmitic acids, which formed 80% of the total number of fatty acids. A notable amount of arachidonic acid, a precursor of eicosanoid hormones, was found in R. acheniorum, R. aurantiaca and R. bacarum. Depending on the strain, 1–10 carotenoid pigments were detected; β-carotene was always the major carotenoid present. Received: 13 December 1994 / Accepted: 18 May 1995     

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Fatty acid composition of the cold-water-inhabiting freshwater red alga Sirodotia Kylin

Four samples of freshwater alga Sirodotia (class Rhodophyceae) collected from two distinct streams in the Mahabaleshwar, Satara district (1,732 m a.s.l.) of the Western Ghats of Maharashtra (India) were analysed for their fatty acid content. The presence of 32 fatty acids was revealed, of which 13 were saturated (SFA), 8 were monounsaturated (MUFA) and 11 were polyunsaturated (PUFA) fatty acids. The major finding was the presence of three pharmaceutically and neutraceutically important PUFAs: arachidonic acid (AA), eicosapentanoeic acid (EPA), and docosahexanoiec acid (DHA). The major fatty acids identified were palmitic (16:0), cis-11,14 icodienoic (20:2), behenic (22:0), cis-8,11,14 eicosatrienoic(20:3n6), cis-4,7,10,13,16,19 docosahexanoeic (22:6n3), cis-13,16 docosadienoic (22:2), erucic (22:1n9), -5,8,11,14,17 eicosapentaenoic (20:5n3), trichosonoic (23:0), nervonic (24:0), arachidonic (20:4n6), cis-10 pentadecanoic (15:1), cis-11,14,17 eicosatrienoic (20:3n3), and myristic acid (14:0). The total PUFA contents ranged from 31.45 to 40.37%. The fatty acids were characterised by the relatively high abundance of PUFAs, while C20 unsaturated acids were appreciably more abundant than C18 unsaturated acids. This is the first report on fatty acid profiles of the genus Sirodotia.     

Fatty acid and lipid composition in mycelia from submerged or surface culture of Streptomyces viridochromogenes

Abstract Streptomyces viridochromogenes was grown both as submerged and surface culture. Mycelia from these cultures were analysed for the composition of lipids and fatty acids. An increase in ornithinolipid content according to incubation time was observed. The addition of phosphate inhibited the ornithinolipid synthesis. A mutant strain with bald phenotype did not exhibit the phosphate inhibition. At the same time, the mutant strain had a higher content of 12-methyltetradecanoic acid.     

Expression of SV40 T antigen in finite life-span hybrids of normal and SV40-transformed fibroblasts

The fusion of normal human fibroblasts with SV40-transformed human fibroblasts resulted in hybrid clones, 85% of which exhibited a finite in vitro life-span. Foci of rapidly dividing cells appeared in 15% of the hybrid clones. The cells within these foci repopulated the culture and could then be subcultured through more than 100 population doublings. One or two foci of dividing cells occurred per culture of 10(5) or more cells. The change to an indefinite life-span was, therefore, a rare event. All hybrid clones, including those that exhibited a finite in vitro life-span, expressed viral T antigen. Thus, even though viral DNA was present and being expressed in all hybrid clones, the senescent phenotype was dominant in these hybrids.     

Trypanosoma cruzi: Fatty acid metabolism in vitro

Trypanosoma cruzi populations, composed primarily of trypomastigote forms, readily converted palmitic acid, linoleic acid, oleic acid, and stearic acid to CO₂. Appreciable amounts of carbon from these four fatty acids were also incorporated into neutral and phospholipid lipids by these parasites. Palmitic acid, a 16 carbon saturated fatty acid, was converted at rates greater than those of the other three fatty acids.     

Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts

Fatty acid components, in both the free and combined form of the intact tropical lichen Teloschistes flavicans, and its isolated photobiont and mycobiont, were analyzed by GC-MS of derived methyl esters. Its rDNA analysis confirmed that the isolated cultured symbionts belong to the genera Trebouxia and Teloschistes, respectively. The fatty acid composition of the lichen did not correspond to those found in the isolated symbionts, suggesting that the fatty acid metabolism is markedly influenced by the symbiosis. Differences in the fatty acid composition in the lichen were observed during the summer (27 degrees C), when the main fatty acids were saturated and in the winter (22 degrees C) when an increase of unsaturated fatty acids occurred. Similar differences of composition were also observed for the cultured mycobiont at different temperatures. The increase in the unsaturation level at low temperatures would maintain the membrane fluidity. Our results are the first on the fatty acids of a tropical lichen and suggest that it is sensitive to small temperature variations, which influences its saturated and unsaturated fatty acid composition.     

Fatty acid composition of triacylglycerols from Wharton's jelly determined by high-performance liquid chromatography

The improved method for HPLC determination of fatty acids was proposed. The chromatographic separation of p-bromophenacyl derivatives of fatty acids under a gradient elution was achieved at 40 °C with an RP-18 LiChroCART 5 column and organic mobile phase containing methanol, acetonitrile, water and TEAP buffer pH 5.6. The quantitative determination of those derivatives was performed at 254 nm. Preeclampsia, the most common pregnancy complication, did not affect triacylglycerol content in the umbilical cord Wharton's jelly in comparison to the control material. However, it changed the composition of fatty acids, bound to that lipid class. The method allows the determination of almost all fatty acids forming the investigated neutral lipid class, contained in a solid tissue sample. The use of TEAP buffer excluded precipitation and flow stoppage in the HPLC system. The method reduced time and costs and might be useful for all other lipid classes and different tissues.