Limnantheoside C (20-hydroxyecdysone 3-O-beta-D-glucopyranosyl-[1-->3]-beta-D-xylopyranoside), a phytoecdysteroid from seeds of Limnanthes alba (limnanthaceae)

A new ecdysteroid glycoside, limnantheoside C (20-hydroxyecdysone 3-O-beta-D-glucopyranosyl-[-->3]-beta-D-xylopyranoside [1]), together with limnantheoside A (20-hydroxyecdysone 3-O-beta-D-xylopyranoside [2]) and 20-hydroxyecdysone (3) have been isolated by bioassay/RIA-directed HPLC analyses of a methanol extract of the seedmeal of Limnanthes alba Hartw. ex Benth. The structure of the novel ecdysteroid glycoside (1) was determined unambiguously by UV, LSIMS and a combination of 1D- and 2D-NMR experiments. These three compounds are isolated from Limnanthes alba for the first time.     

Meeting announcements

The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from [¹⁴C]cholesterol, in five different age groups of male Manduca sexta during pupal-adult development were determined by HPLC. Eight free ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of each ecdysteroid fraction rose sharply by day 4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid. This stage demonstrated high degrees of ecdysone biosynthesis, oxidative catabolism, and phosphorylation. As development proceeded to day 16, total ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was accompanieid by increasing titers of both conjugates and acids resulting from the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were 3-epi-20-hydroxyecdysonoic acid and the phosphate conjugates of 3-epi-20-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone. The ultimate inactivation of the ecdysteroids of M. sexta during pupal-adult development is possibly mediated by two pairs of metabolically-linked processes, one leading to a 3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates.     

Photochemical transformation of 20-hydroxyecdysone: production of monomeric and dimeric ecdysteroid analogues.

Structural modification of 20-hydroxyecdysone (20E) based on photochemical transformation yielded dimeric ecdysteroid 7alphaH,7'alphaH-bis-[(20R,22R)-2beta,3beta,20,22,25-pentahydroxy-5beta-cholest-8(14)-en-6-one-7-yl] as a main product. Its structure was determined by detailed NMR analysis. Furthermore, two new monomeric analogues: 14-epi-20-hydroxyecdysone and 14-deoxy-14,18-cyclo-20-hydroxyecdysone were identified in addition to the earlier described 14-deoxy and 14-hydroperoxy derivatives of 20E. Formation of the specific and so far unique ecdysteroid dimer has not been observed in earlier photo-transformation studies. The transformed dimeric analogue of 20-hydroxyecdysone retained the high agonistic activity on the ecdysone receptor in the B(II)-bioassay compared with the original 20E.     

Affinity labelling of a partially purified ecdysteroid receptor with a bromoacetylated 20-OH-ecdysone derivative

The novel bromoacetyl ecdysteroid IV, (20R,22R)-2 beta,3 beta,14 alpha,20,22,25 xi-hexahydroxy-26-(3- bromoacetoxypropyl)-5 beta-cholest-7-en-6-one, BAEIV, has been synthesized by extending the side chain on C26 of 20-OH-ecdysone. BAEIV meets all the requirements for an affinity-labelling reagent. It reacts with the partially purified ecdysteroid receptors of Drosophila melanogaster rapidly and almost quantitatively. Reactions require only micromolar concentrations of BAEIV. The rate of the affinity-labelling reaction is determined by the association of BAEIV with the ecdysteroid receptor. The value of the apparent reaction rate constant is very similar to that of the association rate constant for the binding of 20-OH-ecdysone to the ecdysteroid receptor. Product analysis of the reaction of [14C]BAEIV with the ecdysteroid receptor revealed two labelled peptides having molecular masses 150 kDa and 90 kDa. The smaller peptide is possibly a proteolytic fragment of the larger peptide. The identification of a 150-kDa peptide by chemical affinity labelling of the ecdysteroid receptor agrees with previously reported photoaffinity-labelling results from our laboratory. The results also demonstrate that the ecdysteroid receptor of D. melanogaster has a molecular mass higher than all other vertebrate steroid hormone receptors studied so far.     

Action of 24-epibrassinolide on a cell line of the beet armyworm, Spodoptera exigua

The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.     

A new ecdysteroid, 2-deoxy-5beta,20-dihydroxyecdysone from the fruits of Diploclisia glaucescens

Chemical investigation of ethyl acetate extract of the fruits of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid 2-deoxy-5beta,20-dihydroxyecdysone, together with 20-hydroxyecdysone, 3-deoxy-1beta,20-dihydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 24-ethyl-20-hydroxyecdysone (makisterone C). Latter two ecdysteroids are reported first time from the family Menispermaceae.     

Metabolism of [3H]-ecdysone in Schistocerca gregaria; formation of ecdysteroid acids together with free and phosphorylated ecdysteroid acetates

The metabolism of [³H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid tit re, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetate, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were identified as metabolites in both the free form and as polar conjugates. Comparison of the intact polar conjugates of the ecdysteroid acetates on two HPLC systems with the corresponding authentic compounds indicated that they were 3-acetylecdysone-2-phosphate and 3-acetyl-20-hydroxyecdysone-2-phosphate. Other major polar metabolites were identified as ecdysonoic acid and 20-hydroxyecdysonoic acid. Ecdysone metabolism in fifth-instar S. gregaria is apparently an age-dependent process. Early in the instar, excretion of both free and conjugated ecdysteroids, as well as ecdysteroid 26-acids, occurs. At this stage the level of ecdysteroid acetates in the conjugated (phosphate) form is high, in contrast to the free ecdysteroids, where ecdysone predominates. When the endogenous hormone titre is high, the formation of ecdysteroid acetates is less, the major excreted matabolites at that stage being conjugated 20-hydroxyecdysone together with ecdysteroid-26-acids, but little free ecdysteroids. Acetylation of ecdysone occurs primarily in the gastric caecae. Ecdysone-3-acetate (mainly as polar conjugate) is also a major product of ingested ecdysone in early fifth-instar Locusta migratoria.     

The ecdysteroids from young embryonated eggs of the tobacco hornworm

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3β-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for an the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxy-ecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3α-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In additon to the six 3β-and 3α-ecdysteroids there were at least an equivalent number of unknown ecdysteroids an of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.     

Anteroposterior gradient during nymphal-adult moulting cycle of the tropical bont tick,Amblyomma variegatum (Acarina: Ixodidae)

Summary The structure of the extensible (alloscutum) and inextensible (scutum) integument of the nymph, Amblyomma variegatum was examined during the whole bloodmeal and the nymphal-adult moulting cycle. Integumental events were tentatively correlated with the ecdysteroid levels measured by radioimmunoassay. We observed that all the integumental events were realised along an anteroposterior gradient. During the 5 days corresponding to the bloodmeal, although the hormone concentration was low, a new endocuticle was deposited on both the alloscutum and scutum. Furthermore, mitoses were initiated in the capitulum. On days 1–2 after the meal, ecdysteroid titres began to increase and reached a first peak corresponding to 4.1 ng 20-hydroxyecdysone equivalents/tick on the 4th day after the ticks dropped off their host. At this time the epidermis of the capitulum was detached and the outline of the adult capitulum was already visible. Mitotic activity in the alloscutum was initiated. On day 6 post-drop, the frontal apolysis was achieved and the ecdysteroid titres declined to basal values. A second peak much higher than the first one (maximum value of 33.7 ng/tick) and identified principally as 20-hydroxyecdysone by HPLC/RIA was noted on the 13th day post-drop. During the period of increase in the ecdysteroid levels (days 9–10 post-drop), the mitotic phase ended in the alloscutum and the apolysis began. Epicuticle was deposited after day 12 postdrop. Then, while the titre fell to low values (about 1.6 ng/tick, days 16–20 post-drop), the exocuticle was deposited and the nymphal cuticle was digested. All adult structures were functional 3 days before ecdysis. In young male as in female adults the mean value of the ecdysteroid levels corresponded to about 2.5 ng/tick. Finally, hydrolysis of tick whole extracts with esterase demonstrated a low increase of RIA-positive material, demonstrating the probable presence of natural ecdysteroid fatty-acid conjugates in this species.     

Biotransformations of 20-hydroxyecdysone and analogues by Curvularia lunata NRRL 2178

20-Hydroxyecdysone, the arthropod moulting hormone, was biotransformed by the fungus Curvularia lunata NRRL 2178 to the rare ecdysteroid, 2-dehydro-3-epi-20-hydroxyecdysone, and the novel 3alpha,9alpha-cyclo ecdysteroid analogue, (20R,22R)-3beta,14alpha,20,22,25-pentahydroxy-3alpha,9alpha-cyclo-5beta-cholest-7-en-2,6-dione in 14 and 44% yields, respectively. Ponasterone A and pterosterone were similarly biotransformed to the corresponding 2-dehydro-3-epi- and 3alpha,9alpha-cyclo-analogues.     

Metabolism of maternal ecdysteroid-22-phosphates in developing embryos of the desert locust,Schistocerca gregaria

The ecdysteroid composition of Schistocerca gregaria eggs at different stages of development was determined by analysis of ecdysteroids labelled maternally from [4-¹⁴C]cholesterol. At all stages studied, highly polar ecdysteroid derivatives predominated, but changes in their composition occurred between day 10 of development and hatching (day 17). During this period, polar conjugates of ecdysone-3-acetate and 3-epi-2-deoxyecdysone appeared together with ecdysteroid acids. At day 17, the polar conjugate of [¹⁴C]ecdysone-3-acetate represented 36% of the total conjugated steroids. Separate in vivo studies on the metabolism of [¹⁴C]ecdysteroid conjugates isolated from newly-laid eggs and consisting primarily of the 22-phosphates of ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone showed that ecdysteroid phosphates could be hydrolysed to give primarily free ecdysone during embryogenesis. Developing eggs can metabolize [³H]ecdysone to ecdysonoic acid, 3-acetylecdysone-2-phosphate and to a lesser extent ecdysone-22-phosphate and 20-hydroxyecdysonoic acid. A polar conjugate of 20-hydroxyecdysone-3-acetate, possibly the 2-phosphate derivative, was detected as a minor metabolite of ecdysone. A scheme of the proposed pathways involved in the metabolism of ecdysteroid-22-phosphates in the developing eggs of S. gregaria is presented.     

天蚕卵黄发生的激素调控

报告了蜕皮激素和保幼激素对天蚕Antheraea yamamai卵黄发生的调控作用。当单独以20－羟基蜕皮酮或保幼激素类似物methoprene处理,以及同时用这两种激素处理天蚕蛹时,蛹期脂肪体和血淋巴中卵黄原蛋白（Vg)含量明显高于对照,即二对Vg的合成起促进作用。然而,卵巢中卵黄蛋白（Vt)含量则因激素种类而异,以保幼激素处理时明显低于对照,以20－羟基蜕皮酮处理则反之,即前抑制卵巢对Vg的摄取,而后则起促进作用。离体培养脂肪体并以激素处理的结果表明,20－羟基蜕皮酮和methoprene均能促进Vg合成,但前作用更。综合考虑上述结果可以认为蜕皮激素对该蚕的卵黄发生起主要调控作用。     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.     

Formation of the A/B cis ring junction of ecdysteroids in the locust, Schistocerca gregaria.

1. The mechanism of formation of the A/B cis ring junction of ecdysteroids in the locust Schistocerca gregaria, was investigated by incorporation of [4-14C,3 alpha-3H], [4-14C,4 alpha-3H] and [4-14C,4 beta-3H]cholesterol into 20-hydroxyecdysone in fifth-instar larvae and into ecdysteroid conjugates in ovaries of maturing adult females. 2. In both systems there was retention of the 4 alpha-3H atom in the ecdysteroid and elimination of the 3 alpha- and 4 beta-3H atoms. 3. The 3H retained in the ecdysone formed from [4 alpha-3H]cholesterol in the ovarian system was probably located at C-4. The results are interpreted by postulating the involvement of a 3-oxo-delta 4 intermediate in ecdysteroid biosynthesis in insects.     

Mode of action of the venom of the ectoparasitic wasp,Euplectrus comstockii, in causing developmental arrest in the European corn borer,Ostrinia nubilalis

Euplectrus comstockii Howard (Hymenoptera: Eulophidae), is an ectoparasitic, gregarious wasp which parasitizes the larval stage of several important lepidopteran pests. Parasitization of both natural and unnatural hosts prevents molting in the parasitized instar. Here we report the effect of wasp venom on the European corn borer (unnatural host), an important pest of corn and other vegetables. Venom collected from venom glands of adultE. comstockii, when injected intoO. nubilalis 5th instars, inhibited the growth rate, development and molting of the injected larvae. The observed effect on molting was dose and age dependent. When 3rd, 4th and 5th instarO. nubilalis were envenomated by adult wasps, the larvae also were developmentally arrested and failed to undergo a molt. However, 3rd and 4th instars underwent apolysis (separation of the epidermis from the old cuticle) and produced new cuticle. Fifth instars did not. A premolt hemolymph ecdysteroid peak was not observed in these experimental 5th instars, but injections of 20-hydroxy-ecdysone induced apolysis and new cuticle formation. Envenomated 4th instars (on becoming pharate 5th instars) exhibited a premolt hemolymph ecdysteroid peak. HPLC/RIA revealed that 20-hydroxyecdysone was present in the hemolymph of these pharate 5th instars. Thus, in the European corn borer, the mode of action of the venom depended upon the instar parasitized. Our results support the presence of a venom component(s) that, in 4th instar hosts, inhibited ecdysis, but did not prevent hemolymph ecdysteroid levels from increasing sufficiently to stimulate apolysis. In 5th instars, the same, or perhaps, a different component(s) ofE. comstockii venom prevented the synthesis/release of ecdysteroid by inhibiting a previously unknown molt-regulating physiological event that occurs between days 3 and 4 of the instar. Deceased     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

Transition from larva to pupa: morphogenesis,cell proliferation and protein synthesis in Bombyx wing disc

Summary

In the present study, an attempt was made to clarify the timing of the transition from proliferation stage to pupal preparation stage of Bombyx wing discs and the correlation of its timing and ecdysteroid release. An histological study was carried out during the period of ecdysteroid increase in hemolymph in the last larval instar. The number of mitoses in wing discs from the feeding stage to the spinning stage was counted. During the feeding stage, the number of mitoses was about 200/disc, with the number increasing up to about 850/disc on the day of the beginning of spinning (S0); it then decreased to about 250/disc at the S1 stage, and finally mitotic figures were absent at S2. Comparing the mitotic number with hemolymph ecdysteroid titer, ecdysteroid during the high mitotic period is close to 100ng/ml, while ecdysteroid during the low mitotic period is about 10ng/ml at day 5 and 500ng/ml at S1, respectively. Two-dimensional gel electrophoresis was carried out to identify the ecdysone-induced polypeptides or the polypeptides synthesized during the spinning stage. Five ecdysteroid-inducible polypeptides were observed during the spinning stage or after 20-hydroxyecdysone (20E) treatment. From these findings, low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation and induce new protein synthesis.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

A new ecdysteroid with unique 9beta-OH and four other ecdysteroids from Silene italica ssp. nemoralis

A new natural ecdysteroid, 9beta,20-dihydroxyecdysone (1) and four related compounds 5alpha-20-hydroxyecdysone (2), 5alpha-2-deoxy-integristerone A (3), integristerone A (4) and 22-deoxy-integristerone A (5) were isolated from the herb of Silene italica ssp. nemoralis. Compound 1 is the C-9 epimer of the known 9alpha,20-dihydroxyecdysone (6) and represents a peculiar steroid skeleton. The structures of the compounds were elucidated by 1D and 2D NMR, IR and MS spectroscopy.     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.