Presence Of 20-hydroxyecdysone Research Articles

Bombyx mori Ecdysone Receptor B1 May Inhibit BmNPV Infection by Triggering Apoptosis.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious threat to sericulture. Nevertheless, no effective control strategy is currently available. The innate immunity of silkworm is critical in the antiviral process. Exploring its molecular mechanism provides theoretical support for the prevention and treatment of BmNPV. Insect hormone receptors play an essential role in regulating host immunity. We found a correlation between Bombyx mori ecdysone receptor B1 (BmEcR-B1) and BmNPV infection, whereas the underlying mechanism remains unclear. In this study, the expression patterns and sequence characteristics of BmEcR-B1 and its isoform, BmEcR-A, were initially analyzed. BmEcR-B1 was found to be more critical than BmEcR-A in silkworm development and responses to BmNPV. Moreover, RNAi and an overexpression in BmN cells showed BmEcR-B1 had antiviral effects in the presence of 20-hydroxyecdysone (20E); Otherwise, it had no antiviral activity. Furthermore, BmEcR-B1 was required for 20E-induced apoptosis, which significantly suppressed virus infection. Finally, feeding 20E had no significant negative impacts on larval growth and the cocoon shell, suggesting the regulation of this pathway has practical value in controlling BmNPV in sericulture. The findings of this study provide important theoretical support for understanding the mechanism of the silkworm innate immune system in response to BmNPV infection.

Low GSK3β activity is required for insect diapause through responding to ROS/AKT signaling and down-regulation of Smad1/EcR/HR3 cascade

Glycogen synthase kinase 3β (GSK3β) plays important roles in gene transcription, metabolism, apoptosis, development, and signal transduction. However, its role in the regulation of pupal diapause remains unclear. In this paper, we find that low GSK3β activity in brains of diapause-destined pupae of Helicoverpa armigera is caused by elevated AKT activity. In response to ROS, AKT phosphorylates GSK3β to decrease its activity. In developing pupal brains, GSK3β can activate the transcription factor Smad1, which binds to the promoter region of the ecdysone receptor (EcR) gene and increases its expression. In the presence of 20-hydroxyecdysone (20E), EcR can bind to USP and increase the expression of 20E-response genes, including HR3, for pupal-adult development. In contrast, high levels of ROS in brains of diapause-destined pupae up-regulate p-AKT, which in turn decreases GSK3β activity. Low GSK3β activity causes low expression of EcR/HR3 via down-regulation of Smad1 activity, leading to diapause initiation. These results suggest that low GSK3β activity plays a key role in pupal diapause via ROS/AKT/GSK3β/Smad/EcR/HR3 signaling.

Application of assisted extraction technologies to optimize the extraction of biologically active substances from plant materials

There is an increased interest in obtaining amaranth grain extracts enriched with biologically active substances by deep processing of raw materials with a further prospect of their use as part of specialized multi-purpose food products. The aim of this work was to evaluate the effectiveness of membrane filtration for the extraction of biologically active substances from amaranth grains. Amaranth grain extract was obtained by enzymatic hydrolysis using a proteolytic enzyme followed by membrane treatment. the developed approach made it possible to obtain an extract from amaranth grains - a source of biologically active peptides, 20-hydroxyecdysone and polyphenols (including saponins). In total, triterpene saponins were concentrated in the extract by 8 times, and the phytoecdysteroid 20-hydroxyecdysone was more than 30 times concentrated. The high content of low molecular weight peptide fractions (more than 80%) in the composition of the extract makes it promising to evaluate its antihypertensive and antidiabetic properties in vivo. The presence of 20-hydroxyecdysone in the extract, an adaptogen with a proven effect, opens up the possibility of its use as a functional food ingredient in specialized food products, including for athletes.

METHOD OF THE RAPID ANALYSIS OF 20-HYDROXYECDYSONE CONTENT IN PLANTS AND FERNS USING SOLID-PHASE EXTRACTION ON POLYAMIDE AND MICROCOLUMN HPLC-UV

A method for the rapid quantitative analysis of 20-hydroxyecdysone in plants was developed using the microcolumn reversed-phase HPLC with UV detection (246 nm) on the ProntoSIL-120-5-C18 column (2×75 mm) and the gradient eluent system LiClO4/HClO4-acetonitrile. The chromatographic stage of the analysis was 5 minutes long, which made it possible to characterize the technique as the fastest. For preliminary purification of the plant extracts, solid phase extraction on polyamide was used which led to an increase in the sensitivity of the analysis. Validation studies showed that the technique characterized by satisfactory metrological data. The values of the limit of detection (LOD) and the limit of quantification (LOQ) of 20-hydroxyecdysone were 3.3 and 10 μg/ml, respectively. The accuracy indices for various levels of 20-hydroxyecdysone content (80–120%) did not exceed 98.57–101.38%. The method applied to the analysis of 20-hydroxyecdysone in 359 species of flowering plants and 12 ferns species growing on the territory of the Buryatia Republic. The presence of 20-hydroxyecdysone was established in 22 species including 18 flowering plants and 4 ferns. The concentration levels of 20-hydroxyecdysone in plants varied from trace (Athyrium filix-femina, Diplazium sibiricum, Pteridium aquilinum) to very high (25.40 mg/g in Rhaponticum uniflorum and 25.87 mg/g in Silene jeniseensis). The presence of 20-hydroxyecdysone was firstly detected in three species including Gastrolychnis gracilis, G. saxatilis and Silene violascens. The developed technique is fast, simple, sensitive and stable and can be recommended for searching purpose to evaluate the new plant sources of 20-hydroxyecdysone.

Target of rapamycin (TOR) controls vitellogenesis via activation of the S6 kinase in the fat body of the tick, Haemaphysalis longicornis

Vitellogenin (Vg) synthesis, vitellogenesis, is an essential process for the development and reproduction of ticks. Our previous finding led to the hypothesis that target of rapamycin (TOR) pathway is important for vitellogenesis in the hard tick, Haemaphysalis longicornis. The TOR pathway controls cellular activity according to nutrient availability in eukaryotes. TOR, a member of the phosphatidylinositol 3-kinase family, is a central player in this pathway. Here, we present preliminary evidence that H. longicornis TOR (HlTOR) controls vitellogenesis via activation of S6 kinase (S6K) in the fat body. RNA interference (RNAi)-mediated gene silencing of HlTOR was undertaken to elucidate the involvement of HlTOR in the vitellogenesis of the tick. HlTOR-RNAi caused inhibition of S6K phosphorylation in the fat body. HlTOR-RNAi also altered not only the expression levels of GATA mRNA and protein but also the intracellular localisation of GATA in the fat body. The expression levels of Vg mRNA and protein in the fat body of HlTOR-RNAi ticks were significantly lower than those in control ticks. In the pre-ovipositional stage, the ovaries of control ticks had brown oocytes developing, but those of HlTOR-RNAi ticks were white and immature. The haemolymph colour indicated that the amount of Vg was lower in HlTOR-RNAi ticks than in the controls. Furthermore, rapamycin inhibited S6K phosphorylation and reduced the expression levels of Vg mRNA and protein in the fat bodies. Vg proteins were not detected in rapamycin-treated fat bodies in the presence of 20-hydroxyecdysone. These results suggest that HlTOR activity is critical for vitellogenesis stimulated by 20-hydroxyecdysone.

Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

In this study, we report the establishment of the binary Gal4/UAS system for the yellow fever mosquito Aedes aegypti. We utilized the 1.8-kb 5′ upstream region of the vitellogenin gene ( Vg) to genetically engineer mosquito lines with the Vg-Gal4 activator and established UAS-EGFP responder transgenic mosquito lines to evaluate the binary Gal4/UAS system. The results show that the Vg-Gal4 driver leads to a high level of tissue-, stage- and sex-specific expression of the EGFP reporter in the fat body of Vg-Gal4/UAS-EGFP hybrids after blood-meal activation. In addition, the applicability of this system to study hormonal regulation of gene expression was demonstrated in in vitro organ culture experiments in which the EGFP reporter was highly activated in isolated fat bodies of previtellogenic Vg-Gal4/UAS-EGFP females incubated in the presence of 20-hydroxyecdysone (20E). Hence, this study has opened the door for further refinement of genetic tools in mosquitoes.

Do European Corn Borer Females Detect and Avoid Laying Eggs in the Presence of 20-Hydroxyecdysone?

European corn borer larvae detect and avoid feeding in the presence of phytoecdysteroids (PEs) such as 20-hydroxyecdysone (20E). Therefore, we hypothesized that females would have taste receptors similar to larvae and avoid laying eggs in the presence of 20E. We found female-specific taste sensilla on the tarsi that respond to 20E at concentrations as low as 10(-6) M, a threshold comparable to that of larvae. However, in choice tests, females laid a similar number of eggs on 20E-treated and on nontreated artificial substrates (filter paper, glass, and nylon), although they spent significantly more time in behavioral sequences related to substrate assessment when 20E was present. In contrast, when given a choice between maize plants (eight leaves) sprayed with 20E or only the solvent, females laid 70% fewer eggs on the treated than on control plants. These observations suggest that other chemical cues of plant origin must be present at the same time as 20E for females to modify their oviposition behavior.

The insect growth regulator insecticide cyromazine causes earlier emergence in Drosophila melanogaster

The insecticide cyromazine, classified as an insect growth regulator, inhibits the growth and development of Diptera. The precise mode of cyromazine action remains unknown. We investigated mortality and developmental time effects after exposing first instar Drosophila melanogaster larvae to cyromazine for 8-h intervals. Significant increases in mortality were only observed for cyromazine exposure within the first 16 h of the first instar stage, suggesting that cyromazine accumulates in the larvae over time and that there is a delay between cyromazine exposure and effect. We observed that exposure to cyromazine during the early first instar stage resulted in earlier eclosion of adults when compared to cyromazine exposure at later first instar stages. The presence of 20-hydroxyecdysone during cyromazine exposure significantly reduced the lethal effect of cyromazine. We raise the possibility that the mode of cyromazine action is related to the development hormone, 20-hydroxyecdysone.

A steroid hormone affects sodium channel expression in Manduca central neurons

Neuronal differentiation is characterized by stereotypical sequences of membrane channel and receptor acquisition. This is regulated by the coordinated interactions of a variety of developmental mechanisms, one of which is the control by steroid hormones. We have used the metamorphosis of the holometabolous insect, Manduca sexta, as a model to study effects of 20-hydroxyecdysone on the maturation of thoracic neuron membrane channel expression. To test for direct hormone action, neurons were dissociated into primary cell culture on the first day of pupal life. In situ hybridization demonstrated that the amount of expression of the acetylcholine receptor alpha subunit, MARA1, was not affected by 20-hydroxyecdysone. Immunocytochemistry with an antibody directed against the SP19 segment of voltage-gated sodium channels revealed no effect of 20-hydroxyecdysone treatment during the first 6 days in culture. SP19 sodium channel protein was evenly distributed along all neurites. In contrast, after 8 days in culture, 20-hydroxyecdysone increased the amount of SP19 protein expression and strongly affected its distribution in differentiating neurons. In the presence of 20-hydroxyecdysone, patches of high densities of SP19 sodium channel protein were found in growth cones close to the base of filopodia. This is a further step toward unraveling the blend of membrane proteins under the control of steroids during the development of the central nervous system of postembryonic Manduca. Our results, taken together with previous studies, indicate that 20-hydroxyecdysone does not affect the expression of potassium membrane current or of the nicotinic acetylcholine receptor but instead regulates the amplitude of the calcium membrane current and the amount and distribution of SP19 sodium channel protein.

Midgut Cell Culture from Larvae of <I>Dendroctonus valens</I> (Coleoptera: Scolytidae)

Isolated cellular nidi stem cells from the anterior and posterior midgut of larvae of the red turpentine beetle, Dendroctonus valens LeConte, proliferate and differentiate in vitro when cultured in RPMI 1640 medium, in the presence of 20-hydroxyecdysone, fat body extract from Zophobas morio (Blanchard) (Coleoptera: Tenebrionidae) pupae, and conditioned medium. Our data suggest that the stem cells did not increase exponentially as in typical cultures. However, the early presence of stem cells in larval cultures indicates that the proliferation process is higher than in cultures from adult D. valens. After 2 wk in culture, up to 10% of cells were observed in various stages of differentiation. The mean duration of midgut cultures was 82 d.

The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae.

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.

New Functions of 20-Hydroxyecdysone in Lipid Peroxidation.

The antioxidizig action of 20-hydroxyecdysone was investigated in several systems in vitro. Changes in the parameters of chemiluminescence in the presence of 20-hydroxyecdysone were found in rat liver mitochondrial fractions. The effect of 20-hydroxyecdysone on methyl linoleate micelles free radical oxidation was studied. The rate of oxygen uptake in methyl linoleate micelles was found to be smaller when 20-hydroxyecdysone was present than at its absence. The action of 20-hydroxyecdysone in the liposomal membranes oxidation was investigated be means of α-tocopherol consumption. A lower rate of α-tocopherol consumption in the oxidation of liposomal membranes was found when 20-hydroxyecdysone was present. The effect of cholesterol on free radical formation in liposomal membranes was investigated. The rate of free-radical formation was smaller when cholesterol was present in the membrane than in membranes free of cholesterol. The α-tocopherol antioxidant effect in the membrane was also lower when cholesterol was present. We show here for the first time that 20-hydroxyecdysone has an antioxidant action in combination with α-tocopherol in membranes with cholesterol overload. Kinetic measurements were revealed the 20-hydroxyecdysone antioxidant effect on free radical reactions in membranes.

Induction of Sarcophaga central nervous system remodeling by 20-hydroxyecdysone in vitro.

Proliferation and apoptosis of neural cells were found to be induced simultaneously when larval brains of Sarcophaga peregrina were cultured in the presence of 20-hydroxyecdysone (20-HE) for 24 h. The locations of proliferating cells and apoptotic cells in the brain hemispheres were different. The morphology of brains exposed to 20-HE for a short period proceeded to change sequentially when culture was continued for 2 days even in the absence of 20-HE. These changes mainly consisted of enlargement of the brain hemispheres and extension of the interval between two hemispheres, which closely paralleled the morphological changes of brains that occur in the early pupal stage, suggesting that ecdysteroid alone is sufficient to induce the remodeling of the central nervous system of holometabolous insects. Synthesis of a protein with a molecular mass of 66 kDa was shown to be selectively repressed when brains were cultured in the presence of 20-HE.

Spruce budworm ( Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning

We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

26-hydroxylation of ecdysteroids is catalyzed by a typical cytochrome P-450-dependent oxidase and related to ecdysteroid resistance in an insect cell line.

The epithelial cell line from the dipteran Chironomus tentans responds to the insect steroid hormone 20-hydroxyecdysone and the non-steroidal analogue tebufenozide by undergoing a morphogenetic and biochemical differentiation program. Long-term culture in the presence of 20-hydroxyecdysone has resulted in the selection of subclones that are resistant to the steroid but respond normally to the non-steroidal analogue. In the present study, several subclones that were resistant to the steroid hormone have been compared with steroid-sensitive subclones with respect to their capability to metabolize 20-hydroxyecdysone. Homogenates of both types of cells, when incubated with 3H-labelled steroid in the presence of NADPH, producecd 20,26-dihydroxyecdysone, which was further metabolized to two compounds, which behaved less polar than 20-hydroxyecdysone on reverse-phase HPLC. Ecdysone, a less-active hormone precursor, provided 26-hydroxyecdysone as the only product. The metabolites were identified by mass spectrometry coupled to HPLC, chromatography with authentic samples, and formation of acetonides. The structure of 20,26-dihydroxyecydsone was confirmed by 1H-NMR. The enzyme responsible for the synthesis of 20,26-dihydroxyecdysone in the Chironomus cell preparations has been characterized as a typical cytochrome P-450-dependent monooxygenase. It was a strictly microsomal enzyme, sensitive to inhibition by carbon monoxide and imidazole/triazole-based fungicides, and required NADPH for maximal activity. NADH could partly replace NADPH. The Michaelis constant (Km) for 20-hydroxyecdysone was 0.96 microM, and the maximal enzyme velocity (Vmax) was 50 pmol substrate metabolized x mg protein(-1) x min(-1). 26-Hydroxylation of 20-hydroxyecdysone was inhibited by ecdysone, an alternative substrate, and by inokosterone, a product analogue, to 50% at 1.4 microM and 0.73 microM, respectively. When various subclones were compared with respect to their in vitro rate of 20-hydroxyecdysone metabolization, those clones known to be resistant to the steroid were 'high metabolizers' (> 70% relative rate), whereas the sensitive clones were 'poor metabolizers' (< 30% relative rate). Hence, it is tempting to conclude that ecdysteroid resistance of the Chironomus cell clones is due to metabolic inactivation of the steroid hormone.

Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor

The Drosophila ecdysone receptor (DmEcR) is a member of the nuclear receptor superfamily; it functions as an obligate heterodimer with another nuclear receptor, DmUSP. EcR homologs have now been cloned from several other insects. We report here that one such homolog, BmEcR from the commercial silkmoth, Bombyx mori, is a functional ecdysone receptor. Upon dimerization with BmCF1, the silkmoth homolog of DmUSP, BmEcR binds the radiolabeled steroid ligand 125I-iodoponasterone A with K d = 1.1 nM, indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an ecdysone response element (EcRE) derived from the heat shock protein 27 ( hsp27) gene promoter of Drosophila; and, as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyecdysone. Finally, BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans-activation of an ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are—at least qualitatively-functionally equivalent.

Purification, characterization, and cDNA cloning of procathepsin L from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly), and its involvement in the differentiation of imaginal discs.

A cysteine proteinase that preferentially hydrolyzes carbobenzyloxy-Phe-Arg-MCA (MCA, methylcoumaryl-7-amide) was purified from the culture medium of NIH-Sape-4 cells. The molecular mass of this enzyme was easily changed from 50 to 35 kDa, without appreciable loss of enzyme activity. From the analysis of its cDNA, this enzyme was concluded to be a procathepsin L of Sarcophaga. Only procathepsin L was found in the medium, while the mature 35-kDa cathepsin L was found exclusively in the cells, indicating that the cells have a mechanism to secrete procathepsin L selectively. An antibody against procathepsin L was found to inhibit the differentiation of imaginal discs cultured in vitro in the presence of 20-hydroxyecdysone. In fact imaginal discs were shown to secrete procathepsin L into the culture medium. These results suggest that procathepsin L is an essential proteinase for differentiation of imaginal discs. Northern blotting analysis revealed that unfertilized eggs contain a significant amount of mRNA for the procathepsin L as a maternal mRNA.

CulturedAedes albopictus mosquito cells synthesize hormone-inducible proteins

To provide a framework for biochemical investigation of ecdysteroid action in Aedes albopictus mosquito cells, we examined the effect of 20-hydroxyecdysone on cell growth and morphology, synthesis of inducible proteins (EIPs), and expression of a transfected gene regulated by a synthetic ecdysteroid response element. When cells were cultured in the continuous presence of 10(-6) M 20-hydroxyecdysone, the rate of growth decreased and subtle changes in cell morphology were observed. In both Aedes aegypti and A. albopictus cells, synthesis of a small number of radiolabeled proteins, which appeared as minor bands on sodium dodecyl sulfate-polyacrylamide gels, was induced by treatment with 20-hydroxyecdysone. On two-dimensional polyacrylamide gels, 11 EIPs, ranging in size from approximately 22 to 52 kDa, were identified in A. albopictus C7-10 cells. Ten inducible proteins were localized in the cytoplasmic fraction; EIP28 and EIP31 were detected in both cytoplasmic and nuclear extracts, and EIP29 was detected only in the nucleus, at a very low level. None of these proteins corresponded to small heat shock proteins, whose genes are 20-hydroxyecdysone-inducible in some Drosophila cell lines. The juvenile hormone analog, methoprene, induced expression of a 25 kDa protein in C7-10 cells. Although 20-hydroxyecdysone sustained the synthesis of this methoprene-inducible protein, synthesis did not occur in the presence of 20-hydroxyecdysone alone. In transfected A. albopictus cells, expression of a recombinant DNA construct containing two tandem synthetic ecdysteroid regulatory elements based on a D. melanogaster small heat shock protein gene was modestly induced by 20-hydroxyecdysone.

Analysis of sterols in plants and cell cultures producing ecdysteroids: I. Chenopodium album

Cell cultures have been obtained in vitro from a plant producing ecdysteroids, Chenopodium album. The composition of free and esterified sterols was compared in plant and cell cultures after purification by TLC and identification by GC/MS. Free sterols were the most abundant. A selectivity of sterol esterification was noted for 4,4-dimethylsterols (except in roots) and Δ7-sterols. The 4-desmethylsterols from leaves, roots and cell cultures of Chenopodium album were a mixture of Δ7-sterols (>61%) and Δ5-sterols (>36%). The Δ7-sterols were 24-ethylcholesta-7,22-dien-3β-ol and 24-ethylcholest-7-en-3β-ol. The isolated Δ5-sterols were sitosterol and stigmasterol. Traces of cholesterol and Δ5,7-sterols were also detected. Our attention was also given to ecdysteroid contents, which showed much lower quantity in cell cultures than in plants, confirming however the presence of 20-hydroxyecdysone.

The fate of the prosegment in the acute-phase and programmed synthesis of sapecin, an antibacterial peptide of the flesh fly (Sarcophaga peregrina).

The nucleotide sequence of sapecin cDNA suggested that this antibacterial peptide of the flesh fly (Sarcophaga peregrina) is produced from preprosapecin by post-translational processing. We examined the production of sapecin and its prosegment by radioimmunoassay under two different physiological conditions in which its gene is activated, assuming that the prosegment has some biological role. Results suggested that the prosegment is degraded selectively during production of sapecin. We also found that imaginal discs synthesize sapecin when cultured in the presence of 20-hydroxyecdysone.