Effects of 12 beticolins, Cercospora beticola toxins, on proliferation of ras-transformed adrenocortical cell.

Aim: To explore different effects of 12 beticolins, Cercospora beticola toxins, on ras-transformed adrenocortical cell growth inhibition and their functional mechanism.

Methods: Beticolin-induced inhibition was measured with survival cell number determined by an automated photocolorimetric method. The penetration of beticolin was examined by confocal microscopy.

A lipid transfer protein binds to a receptor involved in the control of plant defence responses.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites.

Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells.

Phosphorylation of extracellular signal-regulated kinases (ERK1/ERK2) has been implicated in cell proliferation of mammalian cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1/ERK2 phosphorylation, stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGFalpha) in NIH/3T3 cells. We observed that both PMA and TGFalpha induced ERK1/ERK2 phosphorylation within 5 min of stimulation.

Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes.

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding.

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid-cryptogein complex is 1:1.

Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis.

Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium.

Combination of the novel farnesyltransferase inhibitor RPR130401 and the geranylgeranyltransferase-1 inhibitor GGTI-298 disrupts MAP kinase activation and G(1)-S transition in Ki-Ras-overexpressing transformed adrenocortical cells.

To test the Kirsten-Ras (Ki-Ras) alternative prenylation hypothesis in malignant transformation, we used a novel farnesyltransferase inhibitor competitive to farnesyl-pyrophosphate, RPR130401, and a CaaX peptidomimetic geranylgeranyltransferase-1 inhibitor GGTI-298. In Ki-Ras-overexpressing transformed adrenocortical cells, RPR130401 at 1-10 microM inhibited very efficiently the [(3)H]farnesyl but not [(3)H]geranylgeranyl transfer to Ras. However, proliferation of these cells was only slightly sensitive to RPR130401 (IC(50)=30 microM).

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes.

Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal.

Geranylgeranyl as well as farnesyl moiety is transferred to Ras p21 overproduced in adrenocortical cells transformed by c-Ha-rasEJ oncogene.

The ras-transformed newborn rat adrenocortical (RTAC) cells were obtained by transfection with the mutated c-Ha-rasEJ oncogene. They are proliferative and tumorigenic cells characterized by expression of the c-Ha-rasEJ oncogene and overexpression of a wild-type ras oncogene. The overproduced Ras p21 was identified here as Ki-Ras p21 by western blotting using a specific anti-Ki-Ras monoclonal antibody.

Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst.

Suspension-cultured cells of Nicotiana tabacum generated active oxygen species (AOS) when they were treated with the proteinaceous elicitor, cryptogein. This response was blocked by diphenylene iodonium, an inhibitor of the neutrophil NADPH oxidase. When microsomal extracts of tobacco cells were probed with an antibody directed against the human small G protein Rac2, two immunoreactive proteins were detected at 18.

Inhibition of cellular growth and steroid 11 beta-hydroxylation in ras-transformed adrenocortical cells by the fungal toxins beticolins.

The proliferation of GM16 and 4CDT ras-transformed newborn rat adrenocortical (RTAC) cells and Y1 mouse adrenal tumor cells was inhibited by beticolins, the fungal toxins extracted from Cercospora beticola, at submicromolar concentrations in a dose-dependent manner. Inhibitory concentrations for half the maximum inhibition were 150, 75 and 25 nM for beticolin-1 and 230, 150 and 50 nM for beticolin-2 in GM16, 4CDT and Y1 cells respectively. Beticolins strongly inhibited the production of 11 beta-hydroxysteroids on the second and third days of treatment in a dose-dependent manner between 0.

High-performance liquid chromatographic study of the regulation of phospholipid metabolism in cultured adrenocortical cells.

A rapid high-performance liquid chromatographic (HPLC) method for the separation of phospholipids was developed for minute samples of total lipids (ca. 200 micrograms). The method was applied to the study of the phospholipid metabolism in adrenocortical cell cultures.

Effect of rat plasma high density lipoprotein with or without apolipoprotein E on the cholesterol uptake and on the induction of the corticosteroid biosynthetic pathway in newborn rat adrenocortical cell cultures.

High density lipoprotein (HDL) has been shown to induce the cellular accumulation of cholesterol esters and the biosynthetis of 21-hydroxysteroids (corticosteroids) newborn rat adrenocortical cells cultivated in serum-free medium. In order to identify the component(s) of HDL responsible for these effects, we investigated the ability of rat HDL subfractions and HDL with or without apolipoprotein E to deliver cholesterol to cells and to stimulate the steroid biosynthetic pathways in adrenal cultured cells. The total cholesterol uptake from HDL2 was greater than that observed with HDL rich in apolipoprotein E (HDL1 and HDLc).

Aldosterone biosynthesis induced by ACTH and angiotensin II in newborn rat adrenocortical cells transfected by c-EJ-Ha-ras oncogene.

Adrenocortical cells were obtained by fractionated trypsination of newborn rat adrenal glands and transfected with a plasmid containing the EJ/T24-Ha-ras oncogene. Isolation of adhesive cells led to a proliferative cell line with an overexpression of 21 kDa ras protein. These cells incubated with corticosterone or deoxycorticosterone as the precursor produced a high level of 18-hydroxycorticosterone and aldosterone as identified by gas chromatography- mass spectrometry.

Synthesis and gas chromatographic-mass spectrometric analysis of reduced compounds derived from 6 alpha- and 6 beta-hydroxy-11-deoxycorticosterone.

A series of thirty two 6-hydroxylated steroids were synthesized by selective reduction of the 4-5 double bond, the 3-oxo group, and/or the 20-oxo group of 6 alpha- and 6 beta-hydroxyDOC. The different reactions leading to the production of specific isomers are discussed. The gas chromatographic and spectrometric characteristics of the methoxime-trimethylsilyl (MO-TMS) or trimethylsilyl (TMS) derivatives of the isomers obtained are given.

A serum-free medium with or without a carrier protein supports the growth of the Y-1 mouse adrenocortical cell line and its steroidogenic function.

The development of a method for the serum-free culture of the Y-1 mouse adrenocortical tumor cell line has permitted a detailed search for factors regulating cellular growth and steroidogenesis. The serum-free medium (SFM) was made of Ham's F10 basal medium supplemented with free fatty acids adsorbed on albumin. The SFM complemented with calcium, arachidonic acid and cholesterol, i.

Biosynthesis of (20S)-20 alpha-reduced steroids simultaneously with corticosteroids in primary cultures of newborn rat adrenocortical cells stimulated by ACTH.

Newborn rat adrenal cells in primary culture produce corticosteroid hormones and (20S)-20 alpha-reduced progesterone metabolites in amounts which depend on ACTH concentrations and stimulation time. Eight (20S)-20 alpha-reduced progesterone metabolites, including 18-hydroxy-(20S)-20 alpha-dihydroprogesterone, were identified by comparison of their data in high performance liquid chromatography and in gas chromatography-mass spectrometry to those of existing or newly synthesized reference steroids. Quantitative studies of individual steroid biosynthesis were also performed using high performance liquid chromatography and gas chromatography.

Role of albumin for the cholesterol transport and on the steroidogenic pathways in serum-free medium newborn rat adrenocortical cell cultures.

[3,4-13C]cholesterol-albumin complex incubated in serum-free medium allowed to evaluate quantitatively the transfer of cholesterol in newborn rat adrenocortical cultured cells, its accumulation as free cholesterol or cholesterol esters and its transformation into steroids which were also originated (47%) from intracellular unlabelled cholesterol. Increasing concentrations of albumin up to 5 g/l enhanced the production of total steroids but in the meantime decreased the 21-hydroxylated steroid fraction. Internalization of albumin shown by using [methyl-14C]methylated-albumin as a tracer accounted only for a minor part in the cholesterol uptake but strikingly affected the steroidogenic pathways by favoring the reductive metabolism of progesterone over the corticosteroid biosynthesis.

Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium.

In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21-hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used.

Bioconversion of 16 alpha-hydroxyprogesterone to 16 alpha-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture.

Using newborn rat adrenal cells in primary culture, 16 alpha-hydroxyprogesterone was bioconverted into numerous 16 alpha-hydroxylated steroids. The method of analysis of these steroids comprised the association of column and thin-layer chromatography to gas chromatography-mass spectrometry in order to obtain the mass spectra of pure compounds. The identified compounds resulted principally from the enzymatic reactions of 21-hydroxylation 11 beta-hydroxylation and reduction of the 20-oxo and 3-oxo-4-ene groups.

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-alpha-cyclodextrin complex.

Newborn rat adrenocortical cells were successfully cultured in a serum-free carrier protein-free medium (SPFM) by using alpha-cyclodextrin as a cholesterol carrier and have expressed corticosteroid biosynthesis in this medium. A stable inclusion complex of cholesterol-alpha-cyclodextrin with a molar ratio of almost 1 was obtained for a 5 X 10(-5) mol/1 alpha-cyclodextrin concentration. Cell cultures incubated with [4-14C] cholesterol-alpha-cyclodextrin in SPFM produced, under ACTH stimulation, various 14C labeled steroids with a predominance of corticosterone and 18-hydroxy-11-deoxycorticosterone.

Regulation of 11 beta/18-steroid hydroxylation in newborn rat adrenal cells in primary culture.

In newborn rat adrenal cells in primary culture, the level of activity of the 11 beta/18-steroid hydroxylase system involved in the last step of the corticosteroid biosynthesis is increased by ACTH. A parallel study of 11 beta- and 18-hydroxylation showed the same apparent Km values (64 microM) for both hydroxylations. The Vmax values differed: 11.

Bioconversion of 2 alpha-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture.

The bioconversion of 2 alpha-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11 beta-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate.

The role of ACTH in determining the metabolic pathways of deoxycorticosterone by newborn rat adrenal cells in primary culture.

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found.