Studies on growth and steroid formation in tissue cultures of Holarrhena antidysenterica

Callus cultures derived from the hypocotyl of germinated seedlings of the plant, Holarrhena antidysenterica showed an inherent lack of organ-forming ability when grown under the influence of a wide range of exogenous growth factors. Several sterols were isolated from the callus, of which, the predominant ones were identified as cholesterol, 24-methylenecholesterol, 28-isofucosterol, sitosterol and stigmasterol.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Sitosterol biosynthesis in Hordeum vulgare

Excised barley embryos cultured on a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly biosynthesized sterols. Two deuterium atoms were present in 24-methylenecycloartanol, 24-methylenelophenol and campesterol and a maximum of four deuterium atoms were incorporated into 24-ethylidenelophenol, stigmasterol and sitosterol. Mevalonic acid-[2-¹⁴C(4R)4-³H₁] was utilized by the barley embryos to give 28-isofucosterol with a ³H-¹⁴C atomic ratio of 3:5 and stigmasterol and sitosterol with a ³H-¹⁴C atomic ratio of 2:5. 24-Methylenelophenol and 24-ethylidenelophenol were isolated from barley seed and 24-ethylidenelophenol-[2,4-³H₃] was incorporated into sitosterol by barley seedlings. These results show that in the production of sitosterol a 24-ethylidenesterol intermediate is produced and it is suggested that this is isomerized to give a Δ^24,(25) sterol prior to reduction to the saturated C₂₉ sterol side chain.     

Brassinosteroid/Sterol synthesis and plant growth as affected by lka and lkb mutations of Pea

The dwarf pea (Pisum sativum) mutants lka and lkb are brassinosteroid (BR) insensitive and deficient, respectively. The dwarf phenotype of the lkb mutant was rescued to wild type by exogenous application of brassinolide and its biosynthetic precursors. Gas chromatography-mass spectrometry analysis of the endogenous sterols in this mutant revealed that it accumulates 24-methylenecholesterol and isofucosterol but is deficient in their hydrogenated products, campesterol and sitosterol. Feeding experiments using ²H-labeled 24-methylenecholesterol indicated that the lkb mutant is unable to isomerize and/or reduce the Δ²⁴⁽²⁸⁾ double bond. Dwarfism of the lkb mutant is, therefore, due to BR deficiency caused by blocked synthesis of campesterol from 24-methylenecholesterol. The lkb mutation also disrupted sterol composition of the membranes, which, in contrast to those of the wild type, contained isofucosterol as the major sterol and lacked stigmasterol. The lka mutant was not BR deficient, because it accumulated castasterone. Like some gibberellin-insensitive dwarf mutants, overproduction of castasterone in the lka mutant may be ascribed to the lack of a feedback control mechanism due to impaired perception/signal transduction of BRs. The possibility that castasterone is a biologically active BR is discussed.     

Sterol utilization in honey bees fed a synthetic diet: Effects on brood rearing

The dietary sterols, cholesterol, campesterol, sitosterol, stigmasterol and 24-methylenecholesterol, were tested for their ability to support brood rearing in the honey bee, Apis mellifera L., by adding them singly to a chemically-defined worker bee diet. Diet supplemented with 24-methylenecholesterol supported the greatest survival of worker bees, but diet supplemented with either 24-methylenecholesterol or cholesterol supported the production of nearly equivalent amounts of sealed brood and more than any of the other three sterols tested. Diets containing stigmasterol, sitosterol, campesterol, or no supplement produced less sealed brood, in decreasing order.     

Free sterols,steryl esters,glucosides, acylated glucosides and water-soluble complexes in Calendula officinalis

In 3- and 14-day-old seedlings and in the leaves of Calendula officinalis the following sterols were identified: cholestanol, campestanol, stigmastanol, cholest-7-en-3-β-ol, 24-methylcholest-7-en-3β-ol, stigmast-7-en-3β-ol, cholesterol, campesterol, sitosterol, 24-methylcholesta-5,22-dien-3β-ol, 24-methylenecholesterol, stigmasterol and clerosterol. Sitosterol was predominant in young and stigmasterol in old tissues. Young tissues contained relatively more campesterol but in old tissues a C₂₈Δ^5,22 diene was present suggesting transformation of campesterol to its Δ^5,22 analog, similar to that of sitosterol to stigmasterol. All the identified sterols were present as free compounds and also in the steryl esters, glucosides, acylated glucosides and water-soluble complexes. The variations in the amounts of these fractions in the embryo axes and cotyledons of 3- and 14-day-old seedlings and the distribution of individual sterols among the fractions are discussed.     

Sterol utilization in honey bees fed a synthetic diet: Analysis of prepupal sterols

The sterols of prepupal honey bees, Apis mellifera L., from brood reared by workers fed chemically-defined synthetic diets containing cholesterol, campesterol, sitosterol, stigmasterol, 24-methylenecholesterol, or no sterol over a 12-week period were isolated, identified, and quantified. The major sterol present in each prepupal sample was 24-methylenecholesterol, but significant levels of sitosterol and isofucosterol were also present in every case, as was a very small percentage of desmosterol (usually < 1%). This is the first report of isofucosterol being identified in the sterols of the honey bee. A considerably larger percentage of each dietary sterol was found in prepupae reared by workers fed that particular sterol in the diet. This was most dramatic in the case of the cholesterol diet in which case cholesterol content increased to as much as 17.2% of the prepupal sterols, whereas cholesterol had not exceeded 2.2% in samples from other diet regimens. However, stigmasterol comprised no more than 6.3% of the total sterols in any sample from prepupae fed the stigmasterol diet. The preponderance of 24-methylenecholesterol in all prepupae, regardless of the dietary sterol provided to the workers, as well as the lesser quantities of sitosterol and isofucosterol present in all samples, suggest a unique system of utilization and metabolism of these dietary sterols by the worker bees. Apparently they make available to the brood varying amounts of unchanged dietary sterol plus considerable and fairly constant portions of 24-methylenecholesterol, sitosterol, and isofucosterol drawn from their own sterol pools.     

Comparison of sterols of pollens,honeybee workers,and prepupae from field sites

Sterols from pollen collected by foraging honeybees, Apis mellifera L, at seven field sites were compared with the sterols of foraging adults and/or prepupae collected from colonies at each site. Invariably, the composition of prepupal sterols was comparable to that found in previous cage studies using chemically defined diets containing various dietary sterols: 24-methyl-enecholesterol was the major sterol; sitosterol and isofucosterol were present in lesser, but significant amounts; and a trace amount of cholesterol was identified in each sample. This occurred even though some of the pollen sterols contained little 24-methylenecholesterol, sitosterol, or isofucosterol and a preponderance of certain other sterols, such as δ⁷-stigmasten-3β-ol and δ^7,24(28)-campestadien-3β-ol in goldenrod and corn pollens, respectively. Thus the selective transfer and utilization of sterols in honeybees that have been demonstrated in cage studies with artificial diets were also shown to occur under field conditions.     

4-Desmethylsgerols in the seeds of Solanaceae

Thirteen 4-desmethylsterols: cholesterol, 24-methylcholesterol, 24-ethylcholesterol, stigmasterol, 24-methylcholesta-5,24-dienol, 24-ethylcholesta-5,24-dienol, 28-isofucosterol, 24-methylenecholesterol, cholestanol, 24-methylcholestanol, 24-ethylcholestanol, cholest-7-enol and 24-ethylcholest-22-enol, were identified in the seeds of solanaceous plants. The distribution of these 4-demethylsterols in the seeds of eleven plants among seven genera of the Solanaceae family was determined.     

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C₂₈- and C₂₉-Δ²⁴⁽²⁸⁾-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ²⁴⁽²⁵⁾ intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study 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 (23,23,25-²H₃,28-¹³C)-24-methylenecholesterol, followed by ¹³C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ²⁴⁽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. An in vivo assay 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 the Ajuga hairy 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.     

Biosynthesis of makisterone A and 20-hydroxyecdysone from labeled sterols by the honey bee,Apis mellifera

In an effort to determine the sterol precursor(s) of the 28-carbon ecdysteroid, makisterone A, honey bee pupae (13 days post-oviposition) were injected with radiolabeled sterols and subsequently examined for labeled ecdysteroids. High performance liquid chromatography of the pupal extracts revealed that [³H]campesterol was converted to a compound that behaved chromatographically identical to authentic makisterone A, and [¹⁴C]cholesterol was incorporated into a compound chromatographically like 20-hydroxyecdysone. No incorporation of either 24-[³H]methylenecholesterol or [¹⁴C]sitosterol into an ecdysteroid was observed. The neutral sterols of uninjected honey bee pupae contained 49.8% 24-methylenecholesterol on a relative percent basis and, with three other C²⁸ and C²⁹ sterols, accounted for over 99% of the total sterols present.     

The fate of radiolabelled C28 and C29 phytosterols in the honey bee

The fate of radiolabelled campesterol, sitosterol and 24-methylenecholesterol fed in chemically-defined diets to honey bee (Apis mellifera L.) workers was determined. At various intervals, sterols of prepupae, newly emerged adults and queens were analyzed qualitatively, quantitatively and radiochemically and it was determined that there was not sufficient radioactivity associated with cholesterol and/or desmosterol in any of the samples to verify that any of the three C₂₈ and C₂₉ sterols was dealkylated and converted to cholesterol. Similarly, there was no evidence for the conversion of campesterol or sitosterol to 24-methylenecholesterol. It was concluded that the major portion of the sterols incorporated into the tissues of the brood larvae originated from the worker bees used to establish the colony. There is good evidence supporting the premise that the workers can make available sterols from their endogenous pools to the nutrient in the hive and that they can replenish these sterols with those from the artificial diet. The queen is also able to replenish sterols utilized in egg production from those obtained by the workers from the artificial diet, and at the end of nine weeks queens contained more than four times as much sterol, on a ‘μg sterol per g fresh weight’ basis, than was found in fertile queens at the beginning of the test period.     

Sterols of organs involved in brood food production and of royal jelly in honey bees

The sterols of the organs (hypopharyngeal and mandibular glands and honey stomachs) involved in worker and queen honey bee brood food production, royal jelly and intact nurse bees were analyzed to obtain information on the selective transfer of specific sterols from one generation to the next. No appreciable increase in the percentage of 24-methylenecholesterol, relative to the total sterols isolated from intact honey bee (Apis mellifera L.), prepupae or adults, was found in the hypopharyngeal or mandibular glands or the honey stomachs from nurse bees reared in colonies fed a chemically-defined diet supplemented with 24-methylenecholesterol. The sterols of these organs contained higher levels of cholesterol than did the sterols of whole body extracts. The other major sterols, sitosterol and isofucosterol, occurred at relative concentrations comparable to whole body extracts. Also, there were higher levels of cholesterol in the sterols from glandular tissues of nurse bees maintained on pollen and sucrose solution than in sterols isolated from intact insects. In a separate study, royal jelly collected over a 6-day period had much higher relative percentages of 24-methylenecholesterol and lower levels of sitosterol and isofucosterol than did the pollen fed to these colonies. The sterols of nurse bees in the latter study had an intermediate concentration of 24-methylenecholesterol. The significance of these findings relative to the unique selective transfer of specific sterols from the diet or from endogenous sterol pools of the nurse bees from generation to generation in the honey bee is discussed.     

The sterols of normal and male-sterile maize tassels during development

The steroids of normal and male-sterile (Texas type) genotypes of maize were investigated during tassel development. A bioassay for estrogen activity of the normal meiotic and postmeiotic tassels was negative, indicating estrogen activity (estrone equivalent) much less than one ng/g of plant tissue. The sterols found were cholesterol, campesterol, stigmasterol, sitosterol, and probably isofucosterol, stigmast-7-enol, and 24-methylenecholesterol. In the premeiotic, meiotic, and postmeiotic stages of both genotypes between 300 and 400 μg of C₂₈ and C₂₉ free sterols per g tassels (wet wt) were found, the proportions of the sterols being ca 45% sitosterol, 30% stigmasterol, and 13% campesterol, with less than 5% each of the remaining sterols. In all three stages before saponification more free sterols were found in the normal than in the male-sterile tassels. The differences were significant at the 95% level in the meiotic and post-meiotic stages. The amounts of these sterols derived from esters decreased from approximately 140 μg/g in the premeiotic stage to 50 μg/g in the meiotic stage, and to an undetectable amount in the postmeiotic stage. After application of cholesterol-[4-¹⁴C] to the normal and male-sterile maize leaves for 3 days at meiosis, the label was found in the free sterols and steryl esters of the leaves but only in the free sterols of the tassels.     

Stereochemical fate of C-26 and C-27 during the conversion of isofucosterol to sitosterol and of 24-methylenecholesterol to campesterol and dihydrobrassicasterol in Oryza sativa cell cultures

Administration of pro-R-methyl-13C-labeled isofucosterol to cultured cells of Oryza sativa revealed that the pro-R and pro-S methyls at C-25 become the pro-R and pro-S methyls at C-25 of sitosterol, respectively. Similar administration experiments using pro-S-methyl-13C-labeled 24-methylenecholesterol established that the pro-R and pro-S methyls at C-25 of 24-methylenecholesterol become the pro-R and pro-S methyls of campesterol, and the pro-S and pro-R methyls of dihydrobrassicasterol, respectively. These results are compatible with our recently proposed 'syn-SE2' mechanism' for double bond isomerization of delta 24(28) into delta 24(25).     

Sterols: Tritium-labeling and selective oxidations

Evidence is presented which shows that in the preparation of radiolabeled cholesterol and other Δ⁵-sterols via the technique of tritium exchange-labeling of keto steroids and their subsequent conversion to sterol, ³H is introduced primarily into positions 2 and 6 ([2,2,4.6-³H]-cholest-5-en-3β-ol) rather than exclusively at 2 and 4 as the literature claims. The stoppered-vial method using sodium methoxide versus column using basic alumina was the preferred method of labeling. It gave labeled keto steroids of reproducible specific activity indicative of the number of exchangeable protons. Because the initial oxidation step is critical to the preparation of ³H-sterols especially on the small scale, four common oxidants: pyridinium chlorochromate (Corey reagent), silver carbonate on Celite (Fetizon reagent), aluminum isopropoxide (modified Oppenhauer reagent) and chromic acid (Jones reagent) were compared using as starting materials, cholesterol, cholest-8-en-3β-ol, ergosterol, 24-methylenecholesterol, 24(25)-dihydrolanosterol, lanosterol, sitosterol and stigmasterol. The effectiveness of the oxidant was markedly influenced by the molecular features of the sterol.     

Effect of AY-9944 on sterol biosynthesis in suspension cultures of bramble cells

Bramble suspension cultures normally contain Δ⁵ sterols (sitosterol, campesterol, and isofucosterol). When the cells were grown in a medium supplemented with AY-9944, their content of Δ⁵ sterols was greatly decreased and Δ⁸ sterols accumulated. Six Δ⁸ sterols, including three new compounds, (24R)-24-ethyl-5α-cholest-8-en-3β-ol, stigmasta-8,Z-24(28)-dien-3β-ol, and 4α-methyl-stigmasta-8,Z-24(28)-dien-3β-ol, were identified. AY-9944 probably inhibited the Δ⁸→Δ⁷ isomerase. A stable cell line growing permanently in an AY-supplemented medium was obtained.     

Effect of fenarimol on sterol biosynthesis in suspension cultures of bramble cells

Bramble suspension cultures normally contain Δ⁵-sterols (sitosterol, campesterol and isofucosterol). When the cells were grown in a medium supplemented with fenarimol, 14α-methyl sterols accumulated. Eight 14α-methyl sterols, including two new compounds, 4α,14α-dimethyl-stigmasta-8,Z-24(28)-dien-3β-ol and 14α-methyl-stigmasta-8,Z-24(28)-dien-3β-ol, were identified. Fenarimol probably inhibited the 14α-methyl demethylation. Cell lines growing permanently in 2 fenarimol-supplemented medium were obtained.     

Origin of sterols in uredospores of Uromyces phaseoli

The sterols from healthy bean leaves are β-sitosterol, stigmasterol, campesterol and 28-isofucosterol. An additional sterol observed in bean leaves infected with Uromyces phaseoli was identified as 7,(Z)-24(28)-stigmastadien-3β-ol, which is the major sterol of the uredospores of the fungus. The fungus appears to stimulate sterol synthesis, but most of the increased sterol content of infected leaves can be attributed to the sterol of the uredospores.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.