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PubMed1988

Metabolism of cytotoxic hydroxysterols in cultured cells. Chemical characterization of metabolites.

The metabolism of labelled 7 alpha- and 7 beta-hydroxycholesterol was investigated in two lymphoma cell lines (YAC-1, RDM-4), in murine splenocytes and in HTC hepatoma cells. The structures of the metabolites in lymphoma cells were determined as 3 beta-esters of C14-C20 fatty acids by nuclear magnetic resonance and mass spectrometric studies. In hepatoma cells, more polar metabolites of 7 alpha- and 7 beta-hydroxycholesterol were detected whereas, in non-dividing lymphocyte cells, no metabolic transformation occurs. Therefore, metabolic transformation of the hydroxycholesterol is not required for the expression of their activity and the question of the physiological role of the metabolic products is raised.

Animals↗
PubMed1993

Synthesis of potential C27-intermediates in bile acid biosynthesis and their deuterium-labeled analogs.

In connection with studies of alternative pathways in bile acid biosynthesis, potential intermediates in a pathway starting with 27-hydroxylation of cholesterol have been prepared in natural and deuterated forms. Established methods were used to prepare 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid. Clemmensen reduction of kryptogenin in unlabeled and deuterated solvents yielded 27-hydroxy-cholesterol and 16-oxo-5-cholestene-3 beta,27-diol, which were separated by adsorption chromatography on Unisil. The labeled 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid derived from it consisted of molecules with seven (50%), six (20%), and eight (20%) deuterium atoms, and unlabeled molecules were not detected. The acetates of 27-hydroxycholesterol and methyl 3 beta-hydroxy-5-cholestenoate were 7 alpha-hydroxylated in a copper-catalyzed reaction with tert-butylperbenzoate, and the products were purified by chromatography on Unisil. The 7 beta-epimers were obtained as side products. Labeled 3 beta,7 alpha-dihydroxy-5-cholenoic acid was prepared in the same way from 3 beta-hydroxy-5-[2,2,4,4,23-2H5]-cholenoic acid. The 3-oxo-delta 4 analogs of the 3 beta-hydroxy-delta 5 compounds were prepared by oxidation with cholesterol oxidase. The labeled products had the same isotopic composition as the starting materials. Gas chromatographic retention indices and mass spectral characteristics of the trimethylsilyl ether derivatives of the neutral steroids and the methylated acids are given for all compounds.

Bile Acids and Salts↗
PubMed1986

Inhibition of IL-2 secretion and IL-2 receptor appearance of activated lymphocytes pretreated with hydroxylated sterols.

When murine BALB/c splenocytes are pretreated for 2 h with different concentrations of 25-hydroxycholesterol (25-HC) or 7 beta-hydroxycholesterol (7-HC), washed and stimulated either with irradiated C57BL/6 splenocytes or with Con A, IL-2 secretion is inhibited in a dose-dependent way as well as the subsequent cell proliferation. Using the same treatment and stimulation conditions, IL-2 receptor appearance on human T lymphocytes, as characterized by anti-Tac antibody binding, is also inhibited in a dose-dependent way. In contrast, the hydrophilic derivative of 7 beta-hydroxycholesterol, the 3.7 bishemisuccinate sodium salt (7-HC BHS), did not influence any of the 3 tested parameters.

Animals↗
PubMed1995

Glucocorticoids enhance the cholesterol side-chain cleavage activity of ovine adrenocortical mitochondria.

We have shown previously that a chronic treatment with glucocorticoids enhances cAMP- or ACTH-induced steroidogenesis of cultured ovine adrenocortical cells. This effect appears to involve a greater amount of cholesterol in mitochondria. Hence, the present study aimed to define the role of glucocorticoids in cholesterol metabolism by these cells. 2-day-old cultures were exposed to different hormones or inhibitors (10(-6) M ACTH, 10(-5) M metyrapone) for 28-48 h. At the end of the treatment period, the cells were stimulated for 2 h with 10(-3) M 8Br-cAMP, in the presence of 10(-3) M aminoglutethimide (in order to load mitochondria with cholesterol). Mitochondria were then isolated and incubated without or with 100 microM cholesterol either in the presence or absence of 10(-3) M CaCl2, or with 25 microM 22R-hydroxycholesterol. Mitochondria isolated from dexamethasone-treated cells produced consistently more pregnenolone than mitochondria from control cells, suggesting that at least part of the additional cholesterol present in these mitochondria was available for steroidogenesis. However, similar differences were obtained when mitochondria were incubated in the presence of exogenous cholesterol, both with or without calcium, or in the presence of 22R-hydroxycholesterol. Pregnenolone production under these latter conditions was much higher than when endogenous cholesterol was the only substrate. Conversely, metyrapone treatment of the cells resulted in lower production of pregnenolone from 22R-hydroxycholesterol by their mitochondria. Likewise ACTH treatment enhanced pregnenolone production by isolated mitochondria irrespective of the incubation conditions. These effects of dexamethasone and ACTH were not related to higher amounts of adrenodoxin, adrenodoxin reductase or cytochrome P450scc. These results indicate that exposure of ovine adrenocortical cells to glucocorticoids or ACTH enhances their steroidogenic potency not only by increasing the amount of cholesterol available for steroidogenesis but also by enhancing some step(s) involved in the transformation of cholesterol into pregnenolone.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2007

Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice.

Liver X receptors (LXRs) are nuclear receptors that play crucial roles in lipid metabolism in vivo and are activated by oxysterol ligands in vitro. The identity of the ligand that activates LXRs in vivo is uncertain. Here we provide two lines of evidence that oxysterols are LXR ligands in vitro and in vivo. First, overexpression of an oxysterol catabolic enzyme, cholesterol sulfotransferase, inactivates LXR signaling in several cultured mammalian cell lines but does not alter receptor response to the nonsterol agonist T0901317. Adenovirus-mediated expression of the enzyme in mice prevents dietary induction of hepatic LXR target genes by cholesterol but not by T0901317. Second, triple-knockout mice deficient in the biosynthesis of three oxysterol ligands of LXRs, 24S-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, respond to dietary T0901317 by inducing LXR target genes in liver but show impaired responses to dietary cholesterol. We conclude that oxysterols are in vivo ligands for LXR.

Animals↗
PubMed2006

The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus.

BACKGROUND/AIMS: The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point. METHODS: Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR. RESULTS: r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry. CONCLUSIONS: LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.

Adolescent↗
PubMed1997

Localization of sterol 27-hydroxylase immuno-reactivity in human atherosclerotic plaques.

It has recently been shown that extrahepatic cells can eliminate intracellular cholesterol by enzymatic conversion into 27-hydroxy-cholesterol and 3 beta-hydroxy-5-cholestenoic acid. Using immunohistochemical methods, we studied the presence of the enzyme responsible for these conversions, sterol 27-hydroxylase, in human carotid atherosclerotic plaques. All plaques examined were found to contain sterol 27-hydroxylase immuno-reactive cells. While some endothelial cells stained for sterol 27-hydroxylase, the majority of the immunoreactive cells co-localized with macrophages. Accumulation of sterol 27-hydroxylase-positive cells were often observed in macrophage-rich core regions of complicated lesions. High concentrations of 27-hydroxycholesterol were found in plaques, while the concentration in non-atherosclerotic human vessels was lower by two orders of magnitude. The rabbit, which is particularly sensitive to dietary cholesterol and easily develops fatty streaks, had low plasma levels of 27-hydroxycholesterol, 3 ng/ml compared to 150 ng/ml in humans. The concentration of 27-hydroxycholesterol in the atherosclerotic rabbit vessels was also lower compared to human atherosclerotic plaques. The results are consistent with our hypothesis that sterol 27-hydroxylase may be utilized by human macrophages as a defence towards a high cholesterol load. This mechanism may be less important in some other species.

Animals↗
PubMed1999

Hepatic and extrahepatic dehydrogenation/isomerization of 5-cholestene-3 beta,7 alpha-diol: localization of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in pig tissues and subcellular fractions.

Conversion of 5-cholestene-3 beta,7 alpha-diol (7 alpha-hydroxycholesterol) into 7 alpha-hydroxy-4-cholesten-3-one was studied with microsomes from different pig tissues and with liver subcellular fractions. Dehydrogenase/isomerase activity was efficient in microsomes from liver, ovary and lung, but less efficient in microsomes from adrenal gland and kidney. Microsomes from these tissues, with the exception of lung, were also active in dehydrogenation/isomerization of dehydroepiandrosterone and pregnenolone. Inhibition studies were carried out with trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenases active in steroid hormone biosynthesis (C19/C21-dehydrogenases), and a monoclonal antibody raised against a purified hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. The results showed that the C27-dehydrogenase activity in the tissues was not dependent on the C19/C21 dehydrogenases, but was dependent on the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. Liver mitochondria, cytosol and peroxisomes lacked dehydrogenase/isomerase activity towards 7 alpha-hydroxycholesterol when microsomal contamination was taken into account. Immunoblotting experiments with monoclonal antibodies raised against the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase showed immunoreactivity only with protein in liver microsomes. Immunohistochemical studies showed localization of the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in the bile duct epithelium. It is concluded that 7 alpha-hydroxycholesterol is converted into 7 alpha-hydroxy-4-cholesten-3-one by the microsomal 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in liver and extrahepatic tissues.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Synthesis of 7alpha-hydroxy derivatives of regulatory oxysterols.

7alpha-Hydroxy derivatives of oxysterols are of considerable interest because of their possible involvement in regulation of cholesterol metabolism. This paper describes stereoselective syntheses and complete characterization of the 7alpha-hydroxy derivatives of four key oxysterols: 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S), 25-epoxycholesterol.

Bile Acids and Salts↗
PubMed2000

Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols.

A method of assaying hepatic cytochrome P-450, oxysterol 7alpha-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-(3)H]hydroxycholesterol as a substrate and hydroxypropyl-beta-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7alpha,25-dihydroxycholesterol, into 3-oxo-7alpha,25-dihydroxy-4-cholesten by the activity of 3beta-hydroxy-Delta(5)-C(27) steroid oxydoreductase, a microsomal NAD(+) and NADP(+) dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP(+) into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7alpha-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (-33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (-30%) and also by the oxysterols 7alpha-hydroxycholesterol (-21%), 7beta-hydroxycholesterol (-25%) and epicoprostanol (-20%), and by cyclosporin A (-26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.

Animals↗
PubMed2000

Oxysterol biosynthetic enzymes.

Oxysterols, herein defined as derivatives of cholesterol with a hydroxyl group on the side chain, play several roles in lipid metabolism. Members of this class regulate the expression of genes that participate in both sterol and fat metabolism, serve as substrates for the synthesis of bile acids, and are intermediates in the transfer of sterols from the periphery to the liver. Three abundant naturally occurring oxysterols are 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. The cholesterol hydroxylase enzymes that synthesize each of these have been isolated over the last several years and their study has produced insight into the biology of oxysterols. This article focuses on the properties of these enzymes.

Animals↗
PubMed1995

Isolation and quantification of cholesterol oxides in dairy products by selected ion monitoring mass spectrometry.

A method for isolation, detection and quantification of cholesterol oxidation products based on solid phase extraction in combination with preparative HPLC and gas chromatography-mass spectrometry selected ion monitoring has been developed for dairy products. The isolation procedure had a high recovery and artifact formation was minimal, as shown by isotope labelling. The limits of detection ranged from 0.3 to 35 pg/microliters of the isomeric forms of 7-hydroxycholesterol, 20 alpha-hydroxycholesterol, the isomeric forms of cholesterol-5,6-epoxides, cholestanetriol, 25-hydroxycholesterol and 7-ketocholesterol corresponding to a limit of quantification of 2-6 ng oxysterol/g lipid in the dairy product, depending on the nature of the cholesterol oxidation product.

Cholestanols↗
PubMed1981

Enzyme-bound sterols of bovine adrenocortical cytochrome P-450scc.

Bovine adrenocortical cytochrome P-450 capable of cleaving the side chain of cholesterol was purified to homogeneity by the method of Suhara et al. [Suhara, K., Gomi, T., Sato, H., Itagaki, E., Takemori, S., & Katagiri, M. (1978) Arch. Biochem Biophys. 190, 290]. The substrate-bound form of the enzyme preparation was shown to contain in addition to cholesterol (1.2-3.0 mol/mol of P-450) 0.4 mol of (22R)-22-hydroxycholesterol, 0.1 mol of (20R,22R)-20,22-dihydroxycholesterol, and a trace amount (0.005 mol) of (20S)-20-hydroxycholesterol per mol of P-450scc. This relatively large concentration of (22R)-22-hydroxycholesterol is in accord with the hypothesis that the major pathway leading to side-chain cleavage proceeds through initial hydroxylation at the 22 position. The presence of these sterols as native constituents of cytochrome P-450scc supports their role as enzyme-bound intermediates in the biosynthesis of pregnenolone from cholesterol.

Adrenal Cortex↗
PubMed2003

Broad substrate specificity of human cytochrome P450 46A1 which initiates cholesterol degradation in the brain.

The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. This reaction produces biologically active oxysterol, 24(S)-hydroxycholesterol, and is also the first step in enzymatic degradation of cholesterol in the brain. We report here that P450 46A1 can further metabolize 24(S)-hydroxycholesterol, giving 24,25- and 24,27-dihydroxycholesterols in both the cell cultures transfected with P450 46A1 cDNA and the in vitro reconstituted system with recombinant enzyme. In addition, P450 46A1 was able to carry out side chain hydroxylations of two endogenous C27-steroids with and without a double bond between C5-C6 (7alpha-hydroxycholesterol and cholestanol, respectively) and introduce a hydroxyl group on the steroid nucleus of the C21-steroid hormones with the C4-C5 double bond (progesterone and testosterone). Also, P450 46A1 was found to metabolize xenobiotics carrying out dextromethorphan O- and N-demethylations, diclofenac 4'-hydroxylation, and phenacetin O-deethylation. Thus, substrate specificities of P450 46A1 are not limited to cholesterol and include a number of structurally diverse compounds. Activities of P450 46A1 suggest that, in addition to the involvement in cholesterol homeostasis in the brain, this enzyme may participate in metabolism of neurosteroids and drugs that can cross the blood-brain barrier and are targeted to the central nervous system.

Binding Sites↗
PubMed2003

Oxysterol-induced toxicity in R28 and ARPE-19 cells.

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0-50 microg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe-based assay (2',7'-dichlorodihydrofluorescein diacetate [H2DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 micro/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol-treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.

Animals↗
PubMed1993

Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level.

The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.

Animals↗
PubMed1997

Altered regulation of cholesterol and cholesteryl ester synthesis in Chinese-hamster ovary cells overexpressing the oxysterol-binding protein is dependent on the pleckstrin homology domain.

Oxysterol-binding protein (OSBP) is a high-affinity receptor for a variety of oxysterols, such as 25-hydroxycholesterol, that down-regulate cholesterol synthesis and stimulate cholesterol esterification. To examine a potential role for OSBP in regulating cholesterol metabolism, we stably overexpressed this protein in Chinese-hamster ovary (CHO)-K1 cells. Compared with mock-transfected controls, several cell lines overexpressing wild-type OSBP (CHO-OSBP) displayed a 50% decrease in cholesteryl ester synthesis when cultured in medium with delipidated serum, 25-hydroxycholesterol or low-density lipoprotein (LDL). CHO-OSBP cells showed a 40-60% decrease in acyl-CoA:cholesterol acyltransferase activity and mRNA, a 50% elevation in mRNA for three sterol-regulated genes [LDL receptor, 3-hydroxy-3-methylgluraryl (HMG)-CoA reductase and HMG-CoA synthase], and an 80% increase in [14C]acetate incorporation into cholesterol. CHO-K1 cells overexpressing two OSBP mutants with a complete or N-terminal deletion of the pleckstrin homology (PH) domain had cholesterol esterification and synthesis rates that were similar to those shown by mock-transfected controls. Unlike wild-type OSBP, both PH domain mutants displayed diffuse cytoplasmic immunofluorescence staining and did not translocate to the Golgi apparatus in the presence of 25-hydroxycholesterol. CHO-K1 cells overexpressing OSBP have pronounced alterations in cholesterol esterification and synthesis, indicating a potential role for this receptor in cholesterol homoeostasis. The phenotype observed in cells overexpressing OSBP is dependent on the PH domain, which appears to be necessary for ligand-dependent localization of OSBP to the Golgi apparatus.

Animals↗
PubMed1999

Biotransformations of putative phytoecdysteroid biosynthetic precursors in tissue cultures of Polypodium vulgare.

Incubation of calli and prothalli of Polypodium vulgare with different tritium-labelled ecdysteroids has led to modification of some previous assumptions about the biosynthesis of ecdysteroids in plants. Thus, 25-deoxy-20-hydroxyecdysone was transformed efficiently in both tissues into 20-hydroxyecdysone (20E), but no 25-deoxyecdysteroids such as pterosterone and inokosterone were formed. Likewise, incubation of 2-deoxyecdysone (2dE) produced exclusively ecdysone (E) and 20E, indicating a high 2-hydroxylase activity in both tissues, despite calli not producing phytoecdysteroids. This 2-hydroxylation was also evident in the transformation of 2,22-dideoxyecdysone (2,22dE) into 22-deoxyecdysone (22dE). Different ecdysteroids that do not occur in P. vulgare were formed in the incubation of 3-dehydro-2,22,25-trideoxyecdysone (3D2,22,25dE) by 3alpha-reduction and 3beta-reduction and 25-hydroxylation processes. The fact that 22,25-dideoxyecdysone and 22dE were the only 2-hydroxylated products formed in this case suggests that only compounds bearing a 3beta-hydroxyl group are substrates for the 2-hydroxylase. Surprisingly, 22-hydroxylation was never observed with either 2,22dE or 3D2,22,25dE, raising the possibility that it could occur at an early step in the biosynthetic pathway. In this respect, labelled 22R-hydroxycholesterol was efficiently converted into E and 20E, whereas 22S-hydroxycholesterol was not transformed into ecdysteroids, because of its unsuitable configuration at C22. Finally, the conversion of 25-hydroxycholesterol into E and 20E was greatly enhanced after thermal treatment of prothalli which induces the release of previously stored ecdysteroids. Thus, P. vulgare prothalli and calli appear to be particularly suitable models for the study of ecdysteroid biosynthesis and its regulation in plants.

Chromatography, High Pressure Liquid↗