The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid.

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin-resistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (x 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark-green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (x 40) in ABA-sensitivity of root elongation and, to a lesser extent, of ABA-induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co-segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild-type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild-type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous application of brassinosteroid, illustrating the pivotal importance of the BR-related SAX1 gene.

The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J. 18, 303-314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3 beta-hydroxyl, delta 5,6 precursors to 3-oxo, delta 4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroid-dependent growth of seedlings in both light and dark conditions.

Cytosolic protons as secondary messengers in elicitor-induced defence responses.

A variety of early elicitor-induced membrane responses have been described, and their possible role in the generation of second messengers involved in the cascades of events leading to the activation of defence genes is actively investigated. Treatment of tobacco cells with a crude elicitor preparation from Phytophthora megasperma, purified oligouronides and a commercial pectate lyase, induce a common set of membrane reactions similar to those described in a variety of plant material, i.e. efflux of K+, extracellular alkalinization, net Ca2+ uptake and membrane depolarization. In the same conditions the three elicitors stimulate the activity of phenylalanine ammonia-lyase (PAL) and O-diphenol methyltransferase (OMT), two enzymes of the phenylpropanoid pathway. A good correlation between the intensity of the membrane response and the extent of enzyme stimulation has been observed. Cytosolic acidifications have also been measured as a rapid response to the different elicitor preparations used. These results show that plant cells (which usually succeed in counteracting pH-perturbing processes associated with their metabolism, with the transport of solutes or with the effect of various factors from the environment) display significant variation in the concentration of cytosolic protons in specific physiological circumstances, such as the perception of signals inducing defence reactions. Direct evidence that these cytosolic pH changes could be interpreted by plant cells as messages involved in triggering defence responses is provided by experiments showing that artificial acidifications of the cytoplasm lead to a co-ordinated stimulation of PAL and OMT. These results stress the need to explore in more detail the role played by cytoplasmic mechanisms underlying those pH changes.

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.

In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control.

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of beta-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of beta-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating beta-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced beta-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 x 10(-5) molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco beta-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.

Cholesterol and prostaglandin synthesis by cultured human skin fibroblasts in the Alagille syndrome involving paucity of interlobular bile ducts.

Children with Alagille syndrome show high serum cholesterol (15-20 mmol/L). To establish correlation of this unusual level of cholesterol with the regulation of cholesterol metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity and synthesis of cholesterol, fatty acids and acidic steroids from [14C]acetate were determined in cultured skin fibroblasts from 2-3 year old children. Prostaglandin E2 (PGE2) synthesis and nucleic acid synthesis were determined in cells when they were growing in medium containing normal, Alagille or fetal bovine serum. These values were similar to values of controls. HMGR activity was found to be similar in cells of control and children with the syndrome, whether the cells were incubated in lipoprotein-deficient or normal medium. Incorporation of acetate into cholesterol was inhibited to a greater extent by lipoprotein-containing medium in control than in children with the syndrome. Fatty acid synthesis was similar in all conditions. 1-7% of the recovered lipid radioactivity in cells and medium separated as acidic steroids. Serum from a donor patient, when included in the medium, did not affect PGE2 or nucleic acid synthesis compared with normal human or fetal bovine serum. The data suggest that cells of children with Alagille syndrome may have a membrane defect of transfer of cholesterol (LDL receptor defect) leading to excessive cholesterol synthesis. Also, synthesis of acidic steroids (bile acid-like material) and their secretion into the medium occurs in normal fibroblasts and those from children with the syndrome.

Permissive role of n-6 polyunsaturated fatty acids on carbohydrate oxidation in human infant skin fibroblasts: one possible mechanism of their intervention on coronary heart disease and diabetes.

UNLABELLED: Many publications indicate the beneficial effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) in the control of coronary heart disease and diabetes, although the mechanism is not clear. Some of our previous results suggest that, in contrast to other lipids, n-6 PUFAs could have a permissive effect on carbohydrate oxidation. To check this hypothesis, we determined pyruvate dehydrogenase (PDH, decarboxylase: EC 1.2.4.1) activity in infant skin fibroblasts (ISF) incubated 6 hours in the presence of 0.25 mM linoleic (LI) or arachidonic (AR) acid, compared to oleic acid (OL) and control ISF incubated without addition of fatty acids. The four groups of cells were preincubated 36 hours either in the presence of fetal bovine serum (FBS), or in the presence of lipoprotein-deprived serum (LPDS). RESULTS: (1) When the ISF were maintained in the medium containing FBS, the two PUFAs had little inhibitory effect on PDH activity, in contrast with the effect of OL. (2) When the ISF were kept in the lipoprotein-deficient medium, PDH activity was low in controls and in the OL cells, but the addition of LI or AR increased the activity. This suggests the role of n-6 PUFAs in enhancing carbohydrate oxidation, under certain conditions.

Extent of 14C incorporation into cholesterol of infant skin fibroblasts incubated with carboxyl-labeled oleic, -linoleic or -arachidonic acid.

In order to determine the incorporation of C1-14C derived from mono- and poly-unsaturated fatty acids into cholesterol of human cells cultured in exponential phase, infant skin fibroblasts (SF) were used at the 5th passage. On Day 6, the SF were preincubated 36 h in a medium containing 5 per cent lipoprotein-deficient serum, and thereafter [1-14C] oleic, -linoleic or -arachidonic acid-without (OL1, LI1 and AR1 group SF), or with the addition of 0.25 mM cold fatty acids (OL2), LI2 and AR2 group SF). Cholesterol specific radioactivity (SRA) peaked 1 h after, and leveled off afterwards in the OL1, LI1 and AR1 groups. Cholesterol-SRA was relatively low in the other groups, but increased progressively, giving a biphasic response: C1-14C derived from from linoleic and arachidonic acids was actively incorporated into cholesterol during the first hours, as compared to C1-14C derived from oleic acid, but stabilized between 6 and 12 h for the LI2 and AR2 group SF incubation. This result appears to be due to the stimulation of pyruvate decarboxylation, observed elsewhere, and consequently to the dilution of the radioactive units in a large pool of non-labeled acetyl-CoA units derived from glucose, when these SF were incubated with 0.25 mM polyunsaturated fatty acids.

Pyruvate dehydrogenase activity in the liver, brain and adipose-tissue of lipid-deprived developing rats. Effect of minute amounts of polyunsaturated fatty acids.

UNLABELLED: The present experiment was carried out using the following diets: FF, fat-free, and LP in same diet with 0.7% sunflower oil - given to the progeny of females kept on the FF diet since the mating. after 10 mM Mg2+ activation of the PDH phosphatase, and rate of [1-14C[ pyruvate decarboxylation into acetyl-CoA ester units was determined in the liver, brain and adipose-tissue of the pair-fed developing rats. RESULTS: In the male progeny, pyruvate dehydrogenase (PDH) activity was higher (61%) in the LP group livers than in the FF group livers, at the end of the 13 week experiment. Such a difference was not observed in the two group brains up to the 91 days postweaning, but was even larger (94%) between adipose-tissues of the LP and FF groups. In the female progeny kept 12 weeks on the diets, PDH activity in the LP group tissues was also higher than in the FF group tissues: 63% in the liver, 43% in adipose-tissues, and less than 10% in the brain. Therefore, a minute amount of lipids high in linoleic acid appeared to increase PDH activity, and especially in the liver and adipose-tissues of animals kept on a strictly fat-free diet. This stimulation of the PDH activity seems closely related to the phospholipid rehabilitation in the tissues (decrease in the trienoic, tetraenoic acid ratio values).

Cholesterol synthesis in the liver, kidneys and brain after injection of uniformly 14C-labeled oleic or linoleic acid to developing rats.

6-day-old suckling rats, born to females kept on a fat-free diet, were used to determine cholesterol and fatty acid specific radioactivity (SRA) in the liver, kidneys and brain, after injection of 3 microCi uniformly 14C-labeled linoleic acid (ULI) or oleic acid (UOL). 1 h after injection, cholesterol SRA was highest in the liver and kidneys, and then decreased when UOL was injected. Cholesterol SRA peaked 3 h after injection of ULI in liver and kidneys. The delay in appearance of the ULI (over UOL) peak of cholesterol SRA may be due to differences in the rates of oxidation of these two labeled fatty acids into acetyl-CoA ester, according to the structural role of ULI. In the period between 3 and 56 h, cholesterol was more radioactive in the three tissues after injection of ULI than after injection of UOL. The radioactivity of saturated fatty acids was low in the ULI and UOL groups of these very young animals. Therefore, cholesterol synthesis seemed to happen at a faster rate than the other lipid syntheses in liver and kidneys, where the rate of linoleic acid elongation into arachidonic acid was also slower than cholesterol synthesis. Different results were obtained in the brain, where arachidonic acid SRA increased rapidly after ULI injection.

[Cholesterol ester storage disease in children. Comparative biochemistry of hepatocyte and fibroblast cultures]. / Maladie de surcharge à esters du cholestérol chez l'enfant. Etude biochimique comparative de cultures d'hépatocytes et de fibroblastes.

The results of biochemical studies in three children with cholesterol ester storage disease are reported. This rare disease (13 published cases) and the related Wolman's disease are characterised by a deficiency of acid lipase. Affected children mostly present with isolated hepatomegaly. Hepatic cells (one patient) and fibroblasts (two patients) were cultured and cholesterol accumulation measured. Hepatic cells contained more cholesterol than fibroblasts but the enzyme deficiency, assessed by the abnormal degree of esterification was the same in both cell types.

Children sea-blue histiocytosis (2 cases) compared with phospholipidosis induced by 4-4' DET (4-4' p (diethylamino-2-ethoxy phenyl) 3-4 hexane) in rat.

Two cases of children with liver and spleen enlargement are reported. Sea-blue histiocytes and Pick cells were found in both cases in liver, spleen, bone marrow and blood. Further more, lysobisphodphatidic acids were identified in phospholipid analysis of liver biopsies and cultived liver cells. Absence of neurologic involvement at 14 and 18 years fo age suggest a Crocker type C of Niemann-Pick disease, i.e. a not yet well defined entity. Resemblance of these morphological and biochemical abnormalities with certain cases of drug poisoning (especially the well-known intoxication by 4-4' DET) is discussed on the basis of results from experimental studies with this drug in the rat.

[Fetal and neo-natal development of brown adipose tissue in guinea pigs and rats. Feto-maternal or milk transfer of essential fatty acids : lipogenesis and morphology (author's transl)]. / Développement foetal et néo-natal du tissu adipeux brun du cobaye et du rat. Transfert foeto-placentaire ou lacté des acides gras essentiels : lipogenèse et morphologie

Brown adipose tissue (BAT) lipogenesis (fatty acid, glycerol and CO2 synthesis) and its morphology determined by optical microscopy, were studied in guinea pigs and rats during intra-uterine life and during the suckling period. Following the receptor induction and after the commencement of the hormone sensitive adenylate-cyclase/lipase system (i.e. on the 60th day in guinea pigs, on the 20th day in rats), the fetal BAT releases fatty acids (NEFA) and is capable of allowing the non-shivering thermogenesis. When the maternal diet and, consequently, the fetal or neonatal BAT are supplied with considerable linoleic acid, NEFA contain a large proportion of essential fatty acids. In vitro, the greater the linoleic acid concentration in these NEFA, the less inhibited is the lipogenesis from (2-14C) pyruvate. Thus, in periods just preceding or succeeding birth, fatty acid and glycerol synthesis are higher when the feto-maternal and/or the milk supply are enriched in linoleic acid than when they contain a large proportion of endogenous fatty acids. Morphological studies indicate that the adipose cell evolution could be nonidentical in BAT more or less enriched in essential fatty acids. Linoleic enriched BAT (of animals born to females kept on a sunflower oil diet) seemed to be in a healthy physiological state at birth, perhaps due to rapid lipid renewal and synthesis in their membranes. The control BAT (of animals born to females kept on a lard diet) appeared loaded with fats and in a worse conservation state at the same age.

[A possible essential metabolite of linoleic acid: lipoic acid, the universal coenzyme of alpha-keto acid oxidation]. / Un dérivé vraisemblablement essentiel de l'acide linoléique: l'acide lipoïque, coenzyme universel de l'oxydation des acides alpha-cétoniques

The metabolism of [14C-U] linoleci acid (LI) and [14C-U] oleic acid were compared by injecting these fatty acids into growing rats and then homogenizing livers into a three phase system (chloroform/methanol/water). The radioactivities of these phases were equilibrated by extracting them X times with the same solvents. The lipid lower phase was discarded and the analysis was carried out on the evaporated hydroalcoholic upper phase. The residue was extracted again with methanol and hydrolyzed (HCl 6N). The acidic solution was evaporated, treated with HCl/methanol, extracted with chloroform and analysed by thin layer chromatography. One of the most radioactive intermediates detected after injecting LI was purified again and identified as lipoic acid, on the basis of: a. retention time in gas-liquid chromatography; b. Rf in thin layer chromatography; c. molecular weight as determined by mass spectrometry. Thus, the most important fate of essential fatty acids (except for their part in the prostaglandin synthesis and membrane formation) seems to be that of a precursor for this covalently bound alpha keto-acid dehydrogenation coenzyme--the link between lipid and carbohydrate metabolisms.