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.     

Cyclization of squalene-2,3-epoxide to 10α-cucurbita-5,24-dien-3β-ol by microsomes from Cucurbita maxima seedlings

Labelled 10α-cucurbita-5,24-dien-3β-ol was obtained from [3-³H]squalene-2,3-epoxide incubated with microsomes of Cucurbita maxima seedlings. By contrast the lanostane triterpenoids [2-³H]cycloartenol, [2-³H]parkeol and their corresponding derivatives [2,12-³H]11-ketocycloartenol and [2-³H]24,25-dihydro-90α,11a-epoxyparkeol, incubated under the same conditions, gave no rearranged products with a cucurbitane skeleton. These results suggest that biosynthesis of cucurbitane triterpenoids from squalene-2,3-epoxide occurs through direct cyclization without the intermediacy of lanostane-type triterpenoids such as cycloartenol or parkeol. Labelled cycloartenol, α-amyrin, β-amyrin and 24-methylenecycloartanol were also obtained from [3-³H]squalene-2,3-epoxide in the same enzymatic system yielding labelled 10α-cucurbitadienol.     

Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Biosynthesis of withanolides in Acnistus breviflorus: biogenetic relationships among the main withanolides

To excised leaves and 15-day-old seedlings of Acnistus breviflorus sodium [1-¹⁴C]acetate, [2-¹⁴C]mevalonolactone and [¹⁴C-methyl]methionine were administered in separate experiments. From the absolute incorporation values of withaferin A (1), jaborosalactone A (2) and jaborosalactone D (3) isolated at different times after administration of the tracers, it was deduced that compound 2 is a precursor of both 1 and 3 and that the withanolides are later biodegraded to unknown products. Inoculation of [¹⁴C]jaborosalactone A confirmed its transformation into 1 and 3.     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

Observations on the biosynthesis of 24-methylcholesterol and 24-ethylcholesterol by Zea mays

Examination of the sterols of Zea mays shoots has established that the 24-ethylcholesterol is predominately the 24α-epimer, sitosterol, but the 24-methylcholesterol is a mixture of the 24α- and 24β-epimers. After incubation of Z. mays shoots with [2-¹⁴C, (4R)4-³H₁]mevalonic acid the sitosterol had a ³H: ¹⁴C atomic ratio of 2.09:5 which is consistent with previous results indicating that a Δ²⁴⁽²⁵⁾ -sterol is implicated in its biosynthesis. By contrast, the 24α- and 24β-methylcholesterol mixture had a higher ³H: ¹⁴C atomic ratio of 2.82:5. This can be explained by the operation of two routes for the elaboration of the 24-methylcholesterol side chain. One may proceed via Δ²⁴⁽²⁵⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the 24α-methylcholesterol with a ³H: ¹⁴C atomic ratio of 2:5. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³- or a Δ²⁵-sterol intermediate to give the 24β1-methylcholesterol with a ³H: ¹⁴C atomic ratio of 3:5. The proportion of these two labelled compounds in the mixture then determines the observed ³H: ¹⁴C atomic ratio (2.82:5). Some evidence for the formation of a Δ²⁵-compound, cyclolaudenol, by Z. mays shoots was provided by incorporation studies employing either [2-¹⁴C]mevalonic acid or [Me-¹⁴C]methionine as the sterol precursor.     

The enzymatic synthesis of S-adenosyl-l-[2(n)-H]homocysteine

A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-³H]methionine to S-adenosyl-l-[2(n)-³H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-³H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-³H]homocysteine is removed by adsorption to dextran-coated charcoal.     

Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture

The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2 g l^?l FW) with SA at 100 μM in the presence of 0.6 mg l^?l BA and 20 mg l^?l spermidine for 4 h exposure time at the 4th week in 20 ml liquid medium recorded higher withanolides production (withanolides A [8.48 mg g^?l DW], withanolides B [15.47 mg g^?l DW], withaferin A [29.55 mg g^?l DW] and withanone [23.44 mg g^?l DW]), which were 1.14 to 1.18-fold higher than elicitation with MeJ at 100 μM after 5 weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.     

Leaf ontogenic phase-related dynamics of withaferin a and withanone biogenesis in ashwagandha (<Emphasis Type="Italic">Withania somnifera</Emphasis> Dunal.)- an important medicinal herb

Withania somnifera (Dunal), a medicinally important plant, is extensively used in traditional Indian herbal preparations as well as for modern nutraceutical and functional food supplements. Its characteristic phytogenic molecules are modified steroidal lactones called withanolides. Withaferin A is a predominant constituent and is pharmacologically active. Here, we studied the dynamics of biogenesis and accumulation of withaferin A and withanone inWithania leaves at various developmental phases (very young, young, mature, and senescent). HPLC analysis was conducted to determine the amassed quantities of these phytochemicals, while theirde novo biosynthesizing capacity was examined via incorporation studies with a radio-labeled primary precursor, [2-¹⁴C]-acetate. Denovo biogenesis and accumulation of withanolides was most active in young leaves. Here, we also discuss developmental patterns and secondary metabolism in relation to eco-physiology and phytopharmaceutical variability.     

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol–glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera.     

The disposition of [24-3H]cerebrosterol in developing rat brain.

—The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-¹⁴C]cholesterol, [24-³H]cerebrosterol, and [24-³H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-¹⁴C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-³H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-³H]24-epicerebrosterol was essentially the same as that of [24-³H]cerebrosterol. Ratios of radioactivities of [24-³H]cerebrosterol and [4-¹⁴C]cholesterol accentuated the early accumulation of [24-³H]cerebrosterol in myelin, nerve endings, and microsomes, and declining ³H:¹⁴C ratios disclosed the rapid elimination of [24-³H]cerebrosterol and [24-³H]24-epicerebrosterol relative to [4-¹⁴C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-³H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.     

Biosynthesis of cardenolides in Digitalis lanata

[8-³H]-Cholesterol was synthesized. A doubly labelled sample of [8-³H, 4-¹⁴C]-cholesterol was administered to Digitalis lanata plants and the cardenolides were isolated. Biosynthesized digitoxigenin and digoxigenin retained all the tritium. Barring the migration of the tritium in biosynthesis the results are interpreted as indicative that neither intermediates with δ7, δ⁷ or δ⁸⁸⁽¹⁴⁾ are participating in the elaboration of cardenolides.     

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca2+-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-1

We have investigated the metabolism of 1-O-[³H]octadecyl-sn-glycero-3-phosphocholine ([³H]lyso PAF) and [³H]myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-1 (ET-1). When cells were labelled with [³H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE). In contrast, incubation with [³H]myristate labelled mainly PC. Both precursors were incorporated into all PC and PE subclasses. However, [³H]lyso PAF labelled mainly alkyl-subclasses while [³H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-1 induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca²⁺-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-1 stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.     

Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28^th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.     

Sterol biosynthesis via cycloartenol in Saprolegnia

The Oomycete Saprolegnia ferax incorporates ³H from both cycloartenol-[2-³H] and lanosterol-[2-³H] into its normal sterols cholesterol, fucosterol, desmosterol, and 24-methylenecholesterol. It is concluded that sterol biosynthesis in this organism is via cycloartenol and the taxonomic implications are discussed.     

Compartmentation of cytosolic dehydrogenases studied by transfer of tritium from labelled substrates into lactate in rat hepatocytes

This paper describes the transfer of tritium from [2-³H]xylitol or (1R)-[1-³H]ethanol into lactate in cells from fed rats either untreated or triiodothyronine-treated. The labelling pattern of lactate during the metabolism of [2-³H]xylitol or (1R)-[1-³H]ethanol follows the equation $\text{L = K(1?e}{}^{\mathrm{<mtext>?</mtext><mtext>t</mtext><mtext>\tau </mtext>}}\text{)}$ (μmol tritium/μmol lactate). The yield in lactate together with the minimum value of the total flux of reducing equivalents are used to estimate the specific radioactivity of NADH. We have calculated the lactate dehydrogenase-catalysed oxidation rate of NADH from the experimental values of lactate labelling and the specific radioactivity of NADH. We found the calculated flux of reducing equivalents from NADH to pyruvate to be of the same order of magnitude whether labelled ethanol or labelled xylitol was metabolized. We found the flux to be only a few percent of the maximal activity of lactate dehydrogenase. The results obtained suggest that the cytoplasm can be regarded as one compartment, containing a single pool of NAD(H).     

A general procedure for the preparation of labeled l-ascorbic acid from labeled d-glucose

A simple, three-step conversion of 1,2-O-isopropylidene-α-d-glucofuranose into l-ascorbic acid, originally described by Bakke and Theander, was used to prepare l-[4-¹⁴C]ascorbic acid from milligram amounts of d-[3-¹⁴C]glucopyranose in 28% radioisotopic yield. In addition, l-[6-¹⁴C]- and l-[U-¹⁴C]-ascorbic acid were prepared from d-[1-¹⁴C]- and d-[U-¹⁴C]-glucopyranose, respectively. The procedure is useful for the synthesis of l-ascorbic acid bearing isotopic hydrogen, carbon, or oxygen atoms at specific positions, subject only to the availability of starting material.     

Biosynthesis of vitamin E and of the plastoquinones in chloroplasts: Steric course of the decarboxylation

Samples of (3R)- and (3S)-4′hydroxyphenyl[3-²H₁, 3-³H]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto-enol isomerase (tautomerase). 4′-Hydroxyphenyl[3-¹⁴C]pyruvate was obtained by the action of l-amino acid oxidase on dl-[3-¹⁴C]tyrosine, whereas a simple base-catalyzed exchange procedure yielded samples of 4′-hydroxyphenyl[3-³H]- and 4′-hydroxyphenyl[3-²H₂]pyruvate. All labeled samples were converted in situ into the corresponding homogentisic acids on 4′-hydroxyphenyl-pyruvate dioxygenase that is known to catalyze the migration of the acetate side chain with retention of configuration. The isolated doubly labeled homogentisic acids were incubated with chloroplasts from Raphanus sativus cv. saxa Treib, and from the lipophilic products a fraction containing inter alia tocopherol, tocoquinone, and plastoquinone was obtained by chromatographic procedures. The incorporation of radioactivity was between 0.5 and 11% based on homogentisate. Reductive acetylation of the quinones yielded crystalline diacetylhydroquinones, which were submitted to Kuhn-Roth degradation. The radioactive acetate samples thus obtained were analyzed for chirality by an enzymatic procedure previously published. (2R)-[2-²H₁, 2-³H]Homogentisate gave mainly (S)-acetate, whereas (2S)-[2-²H₁, 2-³H]homogentisate was converted mainly into (R)-acetate. It is concluded that the decarboxylation of the side chain occurred with stereochemical retention during the biosynthetic process.     

Action de la flore microbienne du tractus digestif sur la biosynthese de l'acide cholique chez le rat

Axenic and holoxenic (conventional) rats were fed a diet containing trace amounts of [2,4-³H]cholic and [24-¹⁴C]chenodeoxycholic acids. In the feces of both groups of rats, the percentage of labelled bile acids which were ³H-labelled was slightly different. In the experimental conditions used, the intestinal microflora only slightly modified the synthesis of 12α-hydroxylated bile acids.