phytosterols and 24-methylenecholesterol

Topics: Arabidopsis; Brassinosteroids; Cell Membrane; Cell Wall; Cholestanols; Cholesterol; Mutation; Phytosterols; Signal Transduction; Sitosterols; Steroids, Heterocyclic

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ(24(28))-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ(24(25))intermediate. In this study, we characterized theArDWF1gene from an expression sequence tag library ofAjuga reptansvar.atropurpureahairy roots. The gene was functionally expressed in the yeast T21 strain. Thein vivoandin vitrostudy of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-(2)H3,28-(13)C)-24-methylenecholesterol, followed by(13)C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ(24(25))intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes,Oryza sativa DWF1(OsDWF1) was similarly expressed in yeast. Anin vivoassay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in theAjugahairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.

Topics: Ajuga; Amino Acid Sequence; Cholesterol; Molecular Sequence Data; Oxidoreductases; Phylogeny; Phytosterols; Plant Proteins; Sequence Alignment

Gibberellins (GAs) and brassinosteroids (BRs), two growth-promoting phytohormones, regulate many common physiological processes. Their interactions at the molecular level remain unclear. Here, we demonstrate that OsGSR1, a member of the GAST (GA-stimulated transcript) gene family, is induced by GA and repressed by BR. RNA interference (RNAi) transgenic rice plants with reduced OsGSR1 expression show phenotypes similar to plants deficient in BR, including short primary roots, erect leaves and reduced fertility. The OsGSR1 RNAi transgenic rice shows a reduced level of endogenous BR, and the dwarf phenotype could be rescued by the application of brassinolide. The yeast two-hybrid assay revealed that OsGSR1 interacts with DIM/DWF1, an enzyme that catalyzes the conversion from 24-methylenecholesterol to campesterol in BR biosynthesis. These results suggest that OsGSR1 activates BR synthesis by directly regulating a BR biosynthetic enzyme at the post-translational level. Furthermore, OsGSR1 RNAi plants show a reduced sensitivity to GA treatment, an increased expression of the GA biosynthetic gene OsGA20ox2, which is feedback inhibited by GA signaling, and an elevated level of endogenous GA: together, these suggest that OsGSR1 is a positive regulator of GA signaling. These results demonstrate that OsGSR1 plays important roles in both BR and GA pathways, and also mediates an interaction between the two signaling pathways.

Topics: Brassinosteroids; Cholestanols; Cholesterol; Gene Expression Regulation, Plant; Gibberellins; Oryza; Phenotype; Phytosterols; Plant Growth Regulators; Plant Proteins; Plants, Genetically Modified; RNA Interference; RNA, Plant; Steroids, Heterocyclic; Two-Hybrid System Techniques

The evolution of the composition of sterols and squalene during the maturation of the fruit of three cultivars (Achaak, Perlees and Mazetto) almond (Prunus amygdalus Batsh) was investigated. At the same time the evolution of oleic, linoleic and linolenic fatty acids was also studied. The qualitative and quantitative analyses were made by GC-MS and GC-FID. The present study is based on three axes: The first one is the structural and molecular identification of compounds sterolic and squalene, which are based on the principal of cleavage and the fragmentation characteristic of each peak provided by mass spectrometry. The second axis is interested in the physiological phenomenon of phytosterols accumulation: biosynthesis, evolution, and their relation with squalene as well as their interconversion. The third axis is an emergence of a relationship, which seems to exist, between the biosynthetic compounds of the glyceridic fraction of almond oil (mainly fatty acids) and those of the unsaponifiable fraction (particularly sterols). This relation may be established by 24-methylene cholesterol.

Topics: alpha-Linolenic Acid; Cholesterol; Fatty Acids, Unsaturated; Indicators and Reagents; Phytosterols; Plant Oils; Prunus; Seeds; Squalene; Trimethylsilyl Compounds

Sterol composition and content and their seasonal variations over 18 months were investigated in adductor muscle, digestive gland and gonads of Pecten maximus. Sterols were isolated by Silicagel 60 thin layer chromatography and identified by gas chromatography/mass spectrometry. Eleven sterols were identified, with cholesterol, brassicasterol, 24-methylenecholesterol and 22-trans-dehydrocholesterol being the principal components. The same sterols were found in all three tissues independent of season. The relative amounts of each sterol present in each tissue differed. Total sterol levels in gonad and muscle were higher than in digestive gland. Statistically significant differences (P<0.05) were found between the concentrations of each of the sterols isolated from the gonad or muscle and digestive gland. The seasonal variations in the sterol content of the gonad seem be related to the reproductive cycle, while the sterol content of the digestive gland appears to be linked to diet, mainly diatoms or dinoflagellates. The muscle sterol content showed minor changes throughout the year.

Topics: Animals; Cholestadienols; Cholesterol; Chromatography, Thin Layer; Dehydrocholesterols; Digestive System; Gas Chromatography-Mass Spectrometry; Gonads; Isomerism; Mollusca; Muscle, Skeletal; Phytosterols; Seasons; Spain; Sterols

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22alpha-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism.

Topics: Alleles; Amino Acid Sequence; Arabidopsis; Base Sequence; Brassinosteroids; Cholestanols; Cholesterol; DNA Primers; Genes, Plant; Molecular Sequence Data; Mutation; Phytosterols; Plant Proteins; Sequence Homology, Amino Acid; Steroids, Heterocyclic

We have identified the function of the Arabidopsis DIMINUTO/DWARF1 (DIM/DWF1) gene by analyzing the dim mutant, a severe dwarf with greatly reduced fertility. Both the mutant phenotype and gene expression could be rescued by the addition of exogenous brassinolide. Analysis of endogenous sterols demonstrated that dim accumulates 24-methylenecholesterol but is deficient in campesterol, an early precursor of brassinolide. In addition, we show that dim is deficient in brassinosteroids as well. Feeding experiments using deuterium-labeled 24-methylenecholesterol and 24-methyldesmosterol confirmed that DIM/DWF1 is involved in both the isomerization and reduction of the Delta24(28) bond. This conversion is not required in cholesterol biosynthesis in animals but is a key step in the biosynthesis of plant sterols. Transient expression of a green fluorescent protein-DIM/DWF1 fusion protein and biochemical experiments showed that DIM/DWF1 is an integral membrane protein that most probably is associated with the endoplasmic reticulum.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Base Sequence; Brassinosteroids; Cholestanols; Cholesterol; DNA, Plant; Gene Expression Regulation, Plant; Genes, Plant; Membrane Proteins; Molecular Sequence Data; Mutation; Phenotype; Phytosterols; Plant Growth Regulators; Plant Proteins; RNA, Messenger; RNA, Plant; Sequence Homology, Amino Acid; Steroids; Steroids, Heterocyclic; Stigmasterol

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids in Manduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid alpha-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol in Manduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The delta 24-bonds of lanosterol and cycloartenol were effectively reduced by the Manduca delta 24-sterol reductase enzyme, as is the delta 24-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C28 and C29 phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C28 and C29 sterols was readily converted to cholesterol, and a significant amount of 7-dehydrocholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.

Topics: Animals; Cholestadienols; Cholesterol; Ergosterol; Manduca; Phytosterols; Sitosterols; Sterols; Stigmasterol

Quantitative analysis of the local distribution of four noncholesterol sterols, 24-methylene cholesterol, campesterol, stigmasterol, and beta-sitosterol, and of the local distribution of cholesterol in gallstones was performed by mass spectrometry, with D6-cholesterol as an internal standard. The role played by trace amounts of these four noncholesterol sterols in the formation of gallstones was investigated by comparing the amounts of these sterols in different parts of gallstones. It was found that the amounts of the noncholesterol sterols in the inside part were significant greater than the amounts in the outside part of various structural types of gallstones. However, the distribution of the cholesterol did not show such variation. The amounts of noncholesterol sterols distributed locally suggested that these sterols play a role in the formation of gallstones.

Topics: Cholelithiasis; Cholesterol; Female; Gas Chromatography-Mass Spectrometry; Humans; Male; Middle Aged; Phytosterols; Sitosterols; Sterols; Stigmasterol

Cultures of Tetrahymena pyriformis were incubated with various sterols and the extent of dehydrogenation at C-7 and C-22 was determined. The sterols incubated were desmosterol, 22-dehydrodesmosterol, 24-methyldesmosterol, 24 alpha-methylcholesterol (campesterol), 24-methylene-cholesterol, isohalosterol (26,27-bisnorcampesterol, also known as 24,24-dimethylchol-5-en-e beta-ol, a naturally occurring C26-sterol), and 20-isohalosterol. 20-Isohalosterol was not metabolized, while products with delta 7- and delta 22-bonds were formed from isohalosterol and all of the other sterols studied. This confirms an earlier conclusion, based on results with 20-isocholesterol and cholesterol, that inversion of the configuration from 20(R) to 20(S) completely prevents metabolism both in the nucleus and the side chain. On the other hand, changes in the electronics or stereochemistry at C-24 had a direct affect only on metabolism in the side chain. The presence of a methyl group at C-24 reduced the yield of metabolites with a delta 22-bond relative to those with a delta 7-bond producing an accumulation of 7-dehydro metabolite. A double bond at position-24 counteracted this steric effect, presumably by enhancing the rate of dehydrogenation, and a delta 24(28)-bond was more effect than was a delta 24(25)-bond.

Topics: Animals; Cholesterol; Desmosterol; Molecular Conformation; Phytosterols; Sterols; Tetrahymena pyriformis