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. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

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. Ras protein determined by Lowry method were separated by 14 % SDS-PAGE and electroblotted to Immobilon-P transfer membrane and detected with pan-Ras (Ab-3) monoclonal antibody. The Ca2+ chelation by beticolin was investigated using a calcium ionophore. RESULTS: Cell growth inhibition was found dose- and time-dependently at submicromolar level for beticolin-1, -2, and -13 (IC50

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. PMA acts upstream of MEK and via activation of protein kinase C (PKC), as GF109203X, a potent PKC inhibitor, and U0126, a MEK inhibitor, abolished its actions on ERK1/ERK2 phosphorylation. TGFalpha did not act via PKC because GF109203X failed to curtail the degree of ERK1/ERK2 phosphorylation in these cells. DHA alone failed to induce the phosphorylation of these mitogen-activated protein (MAP) kinases; however, this fatty acid significantly curtailed the PMA- but not TGFalpha-induced MAP kinase enzyme activity and phosphorylation in NIH/3T3 cells. Furthermore, we observed that DHA significantly inhibited PMA-induced translocation of two PKC isoforms, PKC alpha and PKC epsilon, from cytosol to plasma membrane. Interestingly, DHA failed to inhibit the PMA-induced translocation PKC delta isoform in these cells. Furthermore, DHA decreased PMA-induced proliferation of NIH/3T3 cells. In this study, we show for the first time that DHA inhibits MAP kinase ERK1/ERK2) activation and proliferation of NIH/3T3 cells via its inhibitory action on PKC alpha and epsilon isoforms.

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. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.

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. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

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. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the 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). GGTI-298 inhibited the growth of these cells with an IC(50) of 11 microM but cell lysis was observed at 15 microM. The combination of 10 microM RPR130401 and 10 microM GGTI-298 inhibited efficiently (80%) cell proliferation. These combined inhibitors but not each inhibitor alone blocked the cell cycle in G(0)/G(1) and disrupted MAP kinase activation. Thus, combination of two inhibitors, at non-cytotoxic concentrations, acting on the farnesyl-pyrophosphate binding site of the farnesyltransferase and the CaaX binding site of the geranylgeranyltransferase-1 respectively is an efficient strategy for disrupting Ki-Ras tumorigenic cell proliferation.

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. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.

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 18.5 and 20.5 kDa. The same experiment performed with cytosolic extracts of tobacco cells led to the observation of a strong immunoreactive protein at 21.5 kDa only in the cryptogein-treated cells. The appearance of this cytosolic protein was related to the production of AOS by the elicited cells. These results provide evidence for the possible involvement of small G proteins, homologous to the neutrophil Rac2 protein, in the regulation of the elicitor-induced oxidative burst in plant.

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. Radioactivity derived from [14C]mevalonolactone was strongly incorporated into Ras p21 overproduced in RTAC cells. RTAC cells pretreated with lovastatin and labeled with either [3H]geranylgeranyl-pyrophosphate or [3H]farnesyl-pyrophosphate incorporated also radioactivity into Ras p21. These results showed that overproduced Ras proteins were geranylgeranylated as well as farnesylated in RTAC cells. These findings suggest that the strategy for inhibiting proliferation of Ki-ras-dependent tumorigenic cells should be directed against not only farnesylation but also geranylgeranylation of Ras p21.

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 0.1 and 1 microM. Beticolins were shown by confocal microscopy to be localized in cytoplasmic organelles about 30-40 min after treatment. This finding favors a direct action of beticolins on mitochondrial steroid 11 beta-hydroxylase albeit another less direct mechanism involving a cytoplasmic signaling pathway cannot be excluded.

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. A complete separation of the different cellular phospholipid classes was achieved in 40 min. Good resolution of the phospholipid peaks was obtained, which allowed the collection of each individual class of phospholipids for further analysis of radioactivity and fatty acid composition by gas chromatography. When cells were incubated with [U-14C]glycerol or [U-14C]palmitate the bulk of the radioactivity was found in cellular phosphatidylcholines. Exogenous phospholipids were incorporated into cellular lipids to a large extent, however without an increase in the cellular phospholipid content. 12-O-Tetradecanoyl-phorbol-13-acetate induced a 20% increase in the polyunsaturated fatty acid content of the cellular phosphatidylethanolamines, but no change was detected in the cellular phosphatidylcholines. The developed method is well-suited to the study of the phospholipid metabolism in adrenocortical cells where the phospholipid metabolism is closely linked to the specialized functions of the cells.

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). Furthermore, the increase of the ratio between 21-hydroxysteroids and reductive metabolites of progesterone was higher with HDL2 than with HDL1 or HDLc. The results of competitive studies between LDL and HDL subfractions indicate that adrenal cells take up cholesterol from HDL2 and LDL by separate mechanisms but that LDL and HDL containing apolipoprotein E share the same uptake processes. In experiments with various concentrations of HDLc or HDL without apolipoprotein E, the adrenal cells displayed a higher affinity for rat HDLc than for rat HDL without apolipoprotein E. However, HDL without apolipoprotein E produced a higher enhancement of the cholesterol cell content and was 3-fold more effective in stimulating 21-hydroxylated steroid production than rat HDLc. Although these findings suggest a participation of HDL with apolipoprotein E in the HDL interaction with rat adrenal cells, the predominant effect on these cells is devoluted to HDL containing mainly apolipoprotein A.

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. ACTH and angiotensin II increased the basal production of aldosterone nineteen-fold and six-fold respectively. Under ACTH stimulation the ratio between aldosterone and 18-hydroxycorticosterone production was 1:3. The transformation of corticosterone under angiotensin II stimulation yielded up to 41% of 18-hydroxycorticosterone (4.7 micrograms/mg of cell protein per 24h) and 4.4% of aldosterone (0.5 microgram/mg of cell protein per 24h) in a low potassium concentration medium (6 mmol/l). To our knowledge this is the first report of continuous proliferative adrenocortical cells producing aldosterone.

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. The gas chromatographic separation of the syn- and anti-isomers of the methoxime in position 3 was found to be characteristic of the configuration of the hydroxyl in position 6. The difference between methylene unit values of syn- and anti- isomers is much larger for the 6 alpha-series than for the 6 beta-series. The mass spectral analysis showed that many ions are specific of the MO-TMS derivatives of steroids with 3,6-dihydroxy-4-ene or 3-oxo-6-hydroxy-4-ene structure. In the case of steroids with a saturated ring A no significant ions characteristic of the presence of a 6-trimethylsilyloxy substituent were found. This work provides previously unavailable reference data on 6-hydroxylated steroids which should facilitate the study of corticosteroid metabolism.

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.e. SFM-S, induced cell proliferation to a density at confluency higher than that obtained with 1% serum-supplemented medium (1%-SSM) and allowed to sustain cell growth for more than six passages. When albumin was replaced by a dextran polymer (Mr = 2 x 10(6)) used as a carrier of lipids instead of albumin (which resulted in a serum-free and protein-free medium, SPFM), the cell number was 75% of that observed with the SFM-S. The addition to SPFM of beta-globulin alone or combined with insulin caused a 2- or 3-fold increase in the final cell density, respectively. The ability of the Y-1 cell line to produce steroids in response to ACTH was found to be higher in SFM-S or SPFM than in 1%-SSM. Furthermore, the addition of beta-globulin to SPFM stimulated steroid hormone biosynthesis with a marked increase in 11 beta-hydroxylated steroid production. These studies demonstrate that the use of a defined mixture of nutriments and of few growth factors permits to sustain not only the cellular proliferation of the Y-1 cell line but also its differentiated function of ACTH-induced steroidogenesis.

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. Several experiments were made without ACTH and with different concentrations of ACTH for periods of more than 3 weeks. The importance of the two main steroidogenic pathways, corticosteroid biosynthesis and progesterone reductive metabolism was modified by ACTH stimulation of the cultured cells. The progesterone reductive metabolism, important without ACTH and in the first days of ACTH stimulation, was decreased by 6.6 mU of ACTH/ml or higher concentrations but remained active throughout the life span of the stimulated cell cultures.

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. Incubation of [4-14C]cholesterol-HDL in serum-free medium compared to those in medium with lipoprotein-deficient serum, in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum-supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20 alpha-reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7-150 micrograms cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways.