商品描述

Product Specification
FORMULA:	C27H44O6
MW:	464.6
CAS NUMBER:	3604-87-3
MERCK INDEX:	13: 3525
PURITY:	≥95%
APPEARANCE:	White to off-white powder.
SOLUBILITY:	Soluble in methanol, 100% ethanol, acetic acid or DMSO. Sparingly soluble in chloroform. Insoluble in water.
SHIPPING:	AMBIENT

General Information

The name ecdysteroids refers to a class of steroid hormones whose main functions are the regulation of ecdysis in arthropods. Ecdysteroid is a generic name for a total of more than 120 different polyhydroxylated steroids present either in plants (phytoecdysteroids), invertebrates (zooecdysteroids), or both. The ecdysteroids are present in all invertebrate phyla and thus represent the most widespread steroid hormones. The molecular weight is between 464 (e.g. ecdysone, ponasterone A) and 480 (20-hydroxyecdysone).

Structure

In contrast to vertebrates, the invertebrates studied so far are unable to synthesise sterols de novo and are therefore dependent on the presence of cholesterol or related sterols. Another difference is the presence of a full side chain as in cholesterol. Zooecdysteroids are therefore mainly C27 steroids, in contrast to the vertebrate steroid hormones (C18, C19, and C21) . All ecdysteroids bear a cis-fused A/B ring junction which is again different from the situation in vertebrates (trans-fused A/B ring junction). A further difference to vertebrate steroid hormones is the good water solubility of ecdysteroids, caused by the high number of hydroxyl groups. Ecdysteroids can occur either in free form or as conjugates. The position of conjugation as well as the main groups which are conjugated with ecdysteroids are shown in .

Like in vertebrate steroid hormone research, nonsteroidal moulting hormone agonists (bisacylhydrazines) are useful tools for the study of hormonal action and for interference with moulting processes. Recently moulting hormone antagonists (cucurbitacins; triterpenoids of plant origin) have also been described.

Physiological Function

In contrast to insects and crustaceans, much less information is available on the physiological roles of ecdysteroids in other phyla. Among parasites there are some data on presence, titre and putative functions available for cestodes and trematodes but most information has accumulated for nematodes. Analogous to arthropods, there are mainly two processes, which are probably regulated by ecdysteroids in nematodes, namely development and reproduction. At least in some nematodes there is a clear correlation between titre of ecdysteroids and the moulting cycle and there is also an effect of ecdysone on reinitiation of meiosis, on differentiation during early embryogenesis, cell rearrangement during gastrulation and on fecundity.

Ecdysteroids have been discovered decades ago as hormones of arthropods; they have effects and moulting and development. There are also several plants containing ecdysteroids. Effects on the mammal organism are discussed, Ecdysteroids are even used for doping purposes.

Therefore we decided to have a closer look on the metabolism of ecdysteroids. This may also help to understand the pharmacodynamics of these hormones better.

These C27 steroids have in common a 7-en-6-one chromophore, sometimes a methyl group at C-24 and several hydroxyl groups increasing their polarity. The structure of ecdysone is given below as an example of these steroids.

They are present both in animals (arthropods) and plants. About 300 species have been identified. They seem to protect plants against most insects. In insects, they are known to trigger a cascade of morphological changes through specific receptors (molting hormones). Relationships between plants and insects have been hypothesized.

Implications

Nonsteroidal moulting hormone agonists induce precocious and incomplete moult and are used for insect pest control. High concentrations of ecdysteroids are deleterious to insects (hyperecdysonism) and also kill nematodes both in vivo and in vitro. Certain amines and amides inhibit phytosterol dealkylation in insects and free-living nematodes and steps in the ecdysteroid biosynthetic pathway in insects and are powerful schistosomicidal agents.

Fig. 1. Ecdysteroids. (A) Structure of ecdysteroids (20-OH-ecdysone, ponasterone A) and primary synthesis products. Arrows indicate preferential sites of conjugation with phosphate, fatty acids, acetate, glucosides, glucuronides and sulfates. (B)Representative of the group of nonsteroidal moulting hormone agonists.

INTRODUCTION

Moulting, development and aspects of reproduction in insects are controlled by the insect moulting hormones (ecdysteroids).

According to the classical scheme, at specific times in immature stages, the prothoracic glands synthesize and secrete ecdysone, which undergoes 20-hydroxylation in several peripheral tissues, yielding the much more active hormone, 20E (20-hydroxyecdysone). The ecdysteroid titre exhibits distinct peaks at specific stages in development. In immature stages, these arise by increased ecdysteroid synthesis in the prothoracic glands; however, in Drosophila pupae, the source appears not to be these glands . Decreases in titre result from enhanced ecdysteroid inactivation reactions, together with possible elevated excretion. A number of transformations contribute to the inactivation of ecdysteroids, including the formation of 3-epi-(3-hydroxy) derivatives, which are regarded as hormonally inactive. Formation of 3-epiecdysteroids, which occurs in many insect orders, including both Lepidoptera and Diptera, entails conversion of ecdysteroid into 3-dehydroecdysteroid, followed by nicotinamide nucleotide-cofactor-dependent irreversible reduction to 3-epiecdysteroid.

These reactions occur with both ecdysone and 20E. EO (ecdysone is the enzyme that catalyses the oxidation of ecdysteroid and was first demonstrated, characterized and extensively purified from the blowfly, Calliphora vicina.

Figure 1 Enzymic interconversions of ecdysone, 3DE and 3-epiecdysone

As a part of our studies aimed at elucidating the regulation of ecdysteroid titre, including the reactions involved in ecdysteroid inactivation, we have characterized EO, 3DE (3-dehydroecdysone) 3-reductase and 3DE 3-reductase in the cotton leafworm, Spodoptera littoralis (Lepidoptera). EO of S. littoralis (SlEO) exhibits peak activity late in the last larval instar, just after the maximum hormone concentration, and thus could be involved in ecdysone inactivation. The enzyme has been purified and its cDNA cloned, with conceptual translation and amino acid sequence analysis, suggesting that it is an FAD flavoprotein which belongs to the GMC (glucose-methanol-choline) oxidoreductase family. Furthermore, Northern blot analysis showed that the EO mRNA transcript is mainly expressed in the midgut, and that fluctuation in the expression during development accounts for the change in the enzyme activity during the instar. Induction by RH-5992, an ecdysone agonist, suggests that SlEO is an ecdysone-responsive gene, whose promoter contains several putative binding motifs for the products of the ecdysone-responsive ‘early genes’, Broad-Complex and FTZ-F1 (Fushi Tarazu-factor 1).

We here report the identification and molecular characterization of EO in the model organism Drosophila melanogaster (DmEO). Although the predicted amino acid sequence of DmEO has a low degree of overall sequence identity with that of SlEO, the amino acid residues predicted to bind substrate are well conserved between the two species. Analysis of expression profiles revealed that the mechanisms of transcriptional regulation of the two enzymes are also very similar.

EXPERIMENTAL

Drosophila strains

Canton S wild-type flies were maintained at 25 °C. Larvae were staged by placing either first or second instar larvae in Petri dishes supplemented with yeast paste and maintaining them at 25 °C. The plates were examined 4 h later for larvae that had moulted. Newly moulted larvae were collected in small vials (Blades Biological, Cowden, Edenbridge, Kent, U.K.) with diet. They were subsequently harvested at 4 h intervals and frozen at -80 °C. Pre-pupae were staged relative to puparium formation, and aged at 4 h intervals for up to 12 h after puparium formation.

Bioinformatics

D. melanogaster homologues of SlEO were sought in FlyBase using BLAST and the resulting sequence set was aligned using T-COFFEE and manipulated further with Jalview. Sequence-clustering analyses were performed using the programs SEQBOOT, PROTDIST, PROTPARS, NEIGHBOR and CONSENSE of the PHYLIP package. Suitable templates for model building of the EOs were sought by a FASTA search among sequences of the Protein Data Bank (PDB) at the European Bioinformatics Institute.

Modelling of residues 24–599 of SlEO and of residues 66–583 of DmEO was performed with MODELLER 6 using the glucose oxidase structures of Aspergillus niger (PDB code 1cf3) and Penicillium amagasakiense (PDB code 1gpe) as templates. No suitable templates for the first 23 residues of SlEO, the first 65 residues of DmEO or the last six residues of DmEO were available. Since the quaternary structure of the GMC class is invariably a dimer, model dimers of the EOs were constructed, with additional MODELLER restraints applied to maintain identical conformation in the two subunits. In the final alignment,

SlEO and DmEO shared 22% and 19–21% sequence identity, respectively, with the templates. Default regimes of model refinement by energy minimization and simulated annealing were employed. Because of the low sequence similarity between target and template, a rigorous iterative modelling protocol was adopted in which 10 models were constructed and analysed for each alignment variant. These models were analysed for packing and solvent-exposure characteristics using PROSA II and for stereochemical properties using PROCHECK. Possible misalignments were highlighted by positive PROSA II peaks, and variations in alignment of these regions examined. When no further improvements could be achieved, the model with the highest PROSA II score was taken as the final model. The PROSA II score of the final model was used to calculate a pG value in order to provide an estimate of model reliability. Protein structures were superimposed using LSQMAN and visualized using O. The structure of ecdysone was obtained by editing of the crystal structure of 20E. Its structure and that of FAD were added to the final SlEO and DmEO models using the predicted Michaelis complex structure of the distantly related cholesterol oxidase as a guide. Diagrammatic representations of the structures were generated using PyMOL . Secondary structures were defined using STRIDE.

RNA isolation

Each collection of animals (30 third-instar larvae or prepupae) was homogenized in 200 l of TRIzol® reagent (Invitrogen). The total RNA was extracted from the homogenate. Each RNA sample was digested by DNase I to eliminate contaminated genomic DNA, and re-purified using the RNeasy Mini Kit (Qiagen).

Quantitative real-time PCR

Real-time PCR was performed with the Opticon 2 thermal cycler (MJ Research, Watertown, MA, U.S.A.) and using the fluorescence dye SYBR® Green I. Total RNA was isolated using RNeasy columns (Qiagen), followed by DNase I treatment using amplification grade DNase-I (Invitrogen) to remove potential contaminating genomic DNA. A sample (2 g) of DNase-I-treated RNA was used as template to synthesize cDNA using SuperScript II reverse transcriptase (Invitrogen).

Real-time PCR conditions consisted of an initial denaturing step at 95 °C for 10 min, followed by 35 cycles of denaturation for 10 s at 94 °C, annealing for 20 s at 60 °C, extension for 15 s at 72 °C, and 75 °C for 1 s to ‘melt off’ any primer-dimer before fluorescence readings were taken. To ensure only the fluorescence signal of the product of interest was detected for c(t) (cycle threshold) calculations, melting curve analysis was performed after each PCR from 65°–95 °C, increasing in 0.2 °C increments, and held for 1 s at each step. DmEO-specific primers used were 5´-CCG ATT CCG ATG ACT ACT GG-3´ (forward) and 5´-CGC TGG CAA TTC CGG CAT AA-3´ (reverse). Drosophila RP49 (ribosomal protein 49)-specific primers used were 5´-CTC ATG CAG AAC CGC GTT TA-3´ (forward) and 5´-ACA AAT GTG TAT TCC GAC CA-3´ (reverse).

Absolute quantification was performed using gene-specific standard curves for each gene. cDNA encoding the open reading frame of DmEO was cloned into the pSI expression vector (Promega). cDNA encoding RP49 was amplified using the gene-specific primers F and R (5´-TGT CCT CAG CTT CAA GAT-3´ and 5´-ATG TTA TCA ATG GTG CTG CTA-3´ respectively), and this was cloned into the pGEM®-T easy vector (Promega). These plasmids were purified and used as templates to construct standard curves ranging from 103–109 copies. All reactions were performed in triplicate.

Expression of Drosophila genes in COS7 cells

The cDNA sequences of 15 candidate genes containing the entire open reading frame was amplified by PCR and cloned into the pSI expression vector. COS7 cells were transfected with the expression vector with or without the cDNA by using LipofectamineTM reagent (Invitrogen). The transfected cells were homogenized at 48 h post-transfection in 50l of 20 mM Tris/HCl, pH 8.0. A 20 l aliquot of this extract was used for enzyme assays.

EO assay

To assay EO activity during Drosophila development, whole bodies from 15 larvae, pre-pupa or pupa were homogenized in 20 mM Tris/HCl, pH 7.0. The homogenate was centrifuged at 17000  for 10 min. The supernatant was assayed by incubation for 3 h at 37 °C in 50 l of assay mixture consisting of 0.1 M sodium phosphate buffer, pH 6.6, and [3H]ecdysone (18.5 kBq; 1.85 TBq/mmol). Assays were quenched with 50 l of methanol, and proteins were removed by centrifugation. Ecdysteroids in the supernatant were analysed by HPLC using a C18 Nova-Pak cartridge (10 cm×5 mm; Waters Associates) on a Waters instrument (Waters Associates) linked to a 440 UV detector set at 254 nm, and eluted with an isocratic solvent system consisting of acetonitrile/0.1% (v/v) trifluoroacetic acid in water (22:78, v/v) at 1 ml/min. Three independent experiments were performed for each assay, with all assays performed in duplicate.

In a similar manner, to assay the recombinant EO activity, conversion of [3H]ecdysone into [3H]3DE was measured 2 days after the transfection of COS7 cells with the candidate gene expression construct. The transfected cells (approx. 2×106 cells) were homogenized in 100 l of 20 mM Tris/HCl, pH 8.0. The homogenates were centrifuged at 17000  for 10 min. The supernatant was assayed as above, except that incubation was for only 1 h at 37 °C.

Feeding of insects with RH-0345

Synchronous larvae of the temperature-sensitive mutant ecd-1 (ecdysoneless-1), which shows reduced ecdysteroid synthesis and metabolism, were allowed to develop at 20 °C until the beginning of the third larval stage, when they were transferred and maintained at 29 °C for 72 h, after which they were subjected to hormone treatments by feeding. Groups of 30 larvae were subjected to RH-0345 (5 mg/ml diet), an ecdysone agonist, or none (control) supplemented food. After 3 h at 29 °C, total RNA was extracted and analysed.

RESULTS

Bioinformatic prediction of the D. melanogaster homologue of SlEO

A BLAST search showed that 15 sequences in the D. melanogaster genome bore clear similarity to SlEO. The e-values associated with the hits ranged from 2e-75 to 7e-38. Seven of the hits had e-values of less than e-70, showing that there was no single clear EO candidate in D. melanogaster. When aligned, all 15 D. melanogaster sequences shared pairwise sequence identities with SlEO in the range 26–32%. Mindful of the fact that the top BLAST hit is by no means always the nearest neighbour in evolutionary terms, and that sequence clustering is increasingly recognized as an important aid to functional annotation (the ‘phylogenomic’ approach;), relationships between the D. melanogaster sequences and SlEO were explored. As shown in

igure 3 Sequence clustering tree of SlEO and its possible D. melanogasterhomologues

The tree was calculated from distance matrix data using the PHYLIP package [18]. Selected bootstrapping confidence values from analysis of 100 replicates are indicated.

the 15 candidate D. melanogaster sequences cluster reliably into three groups. The bootstrapping value associated with this division is 94 for 100 replicates in the tree derived from distance matrix analysis, and 90 for the equivalent tree obtained using maximum parsimony analysis (results not shown). SlEO localizes within the branch also containing the D. melanogaster sequences CG6728, CG9504, CG9509 and CG9512. This analysis appeared to narrow the search for the DmEO down to four candidates, but further inference was unreliable, since the structures of the SlEO-containing branches of the distance matrix and maximum parsimony trees differed (results not shown).

As recognized by the current wave of structural genomics projects, knowledge of protein structure is an important aid to function prediction. This approach extends to carefully constructed model structures. We therefore examined whether a model of SlEO could yield insights into the determinants of ecdysone specificity that would enable a prediction to be made of which D. melanogaster gene sequence encoded the EO. To this end, a model of SlEO was constructed using a rigorous, iterative modelling procedure. The templates were chosen based on the results of a FASTA search of the sequences of known structures deposited in the PDB. The two top hits were glucose oxidases from two yeasts, P. amagasakiense and A. niger, which scored e-values of 7e-13 and 4e-11 respectively. The third ranking hit was almond hydroxynitrile lyase, with an e-value of 7e-8, followed by Phanerochaete chrysosporium cellobiose dehydrogenase, with e-value 1e-5. On the basis of these results, only the glucose oxidases were deemed worthy of use as templates. Interestingly, bacterial cholesterol oxidases, also of known structure, were absent from the FASTA results, showing that substrate similarity is not a useful guide to evolutionary relatedness in the diverse GMC oxidoreductase superfamily. Initial models contained regions with unfavourable positive PROSA II profiles, several of which could be improved through local alignment changes. The monomer of the final model scored -8.32 by PROSA II analysis, corresponding to a perfect pG value of 1.0 and indicative of a largely accurate target-template alignment. The final model also has good stereochemistry; 88% of residues are in the core regions of the Ramachandran plot, and none are in disallowed regions.

A molecule of ecdysone (modelled on the crystal structure of 20E) was docked into the final SlEO model using as a guide the predicted Michaelis complex of cholesterol oxidase. Although this is a distant SlEO relative in the GMC oxidoreductase superfamily (see above), the plausibility of the resultant bound conformation was confirmed by an absence of significant steric clashes and the observation that the hydrophobic steroid ring was favourably accommodated in a largely hydrophobic pocket. While the rigidity of the steroid ring facilitated its placement, it was apparent that diverse conformations for the flexible side chain were possible. We therefore confined our predictions of potential contributors to ecdysone specificity to residues neighbouring the steroid ring. These were Leu96, Met346, Ala446, Leu534 and Trp537 (shown boxed in Figure 2). The first four of these are different in the glucose oxidases, being represented respectively by the more hydrophilic residues tyrosine, threonine, aspartate and arginine, only the last being reasonably conserved in both glucose and EOs. As predicted ecdysone-binding residues, the expectation was that the D. melanogaster sequence encoding EO would contain similar residues to SlEO at these positions. Among the four D. melanogaster sequences in the branch of the sequence-clustering tree also containing SlEO, the analysis highlighted CG9504 in which the five positions were occupied respectively by Leu138, Met377, Met484, Val574 and Trp577. The other D. melanogaster sequences conserved one or more of these positions, but contained a number of significantly different residues. Thus CG6728 has glutamine, asparagine and histidine at the first, second and fifth positions, and CG9512 has threonine, serine, tyrosine and threonine at the first, second, third and fourth positions. The CG9509 residues conform more closely to the expected pattern: leucine, leucine, phenylalanine, valine and tyrosine. We therefore made the prediction that CG9504 was most likely to represent the DmEO, with CG9509 as a potential alternative candidate. Of the initial set of 15 DmEO candidates identified by sequence-clustering analysis, only CG9504, CG9509 and three more contained hydrophobic residues at all five positions. Interestingly, Flybase suggests a choline dehydrogenase activity for the products of seven genes, a glucose dehydrogenase activity for those of four genes and an unknown activity for those of four genes including CG9504.

Function

Sheding skin the hormone is at with the Ajuga turkestanica, separate in the plants such as the leuzea carthamoides, the Cyanotix vaga and the cyanotis arachnoidea etc.s.Was used for the athlete body to have already increased the muscle egg white to synthesize to recover speed to obtain strong muscle power with improvement in early days.

Sheding skin the hormone has to become the egg white chain through a sour assemble of the increment An Ji obviously, stimulating the muscle cell cell quality thus to synthesize in the protein of ability, and that ability can trace back to to the protein a growth of conversion and migration process.Shed skin hormone not only be good for health and security, it contributes to stability at the sebum Chun hurt of cell, make the energy synthesize a step to turn(the atp and the muscle ammonia are sour) normally and improve the liver function to make the organic physique quick orientation environment and pressure change thus.Will shed skin the hormone usage in include an improvement work ability, immune system, weight to lead low run off with fat etc., several aspect problems have already had a report as well.

1.     Increasing the protein synthesizes to adjust positive nitrogen balance

The research manifestation that carried on in the Soviets in 1988 shed skin hormone to contribute to increase the liver and gall protein to synthesize to settle to promote immediately positive nitrogen balance, it caused the muscle quantity rise.Sheding skin the hormone can reduce the machine urea inside the body cool-headed and passes increment to is called the step that the red corpuscle divides to raise the development of the mature red corpuscle cell that the hemoglobin and the hemoglobin cell divide.It causes the incitement to synthesize the metabolism to the protein metabolism, and immediately positive nitrogen inside the body is equilibrium.

2.     Increase the muscle quantity to reduce body fat in the meantime

Placebo, protein with shed skin the hormone experiment to prove as a result that taking in 78 men and women's athletes that temper through hardships through high strength is protein, at 10- days take after show the slight muscle quantity increment.And take the community of the placebo to appear slight muscle quantity to descend;And take to join protein of sheding skin the hormone of, display to have 6-7% muscular tissue quantity risings and 10% fat quantities descend.Safe test carried on in the meantime, show hormone balance to have no difference.

3.     Increase the last long power, the endurance power and energy

117 ages the athlete of in 18~28 years oldly carries on the ability of exercising, weight, the lung function and the carbon dioxide whole line test.All all inhale like the O2 biggest be worth and exhale CO 2:00 one rising.It equals the decrease instauration time, the optimization function, allow the best muscle to synthesize metabolism to reduce fat and the biggest legal power.Equally mean the usage sheds skin the athlete and the usage placebo of the hormone to compare the increment that can experience personally the last long power, the endurance power and energy.And the less is tired and have a better ability, stronger motive, faster speed and strengthen of strength.

Make use of in the hairdressing

Sheding skin the hormone nature changes a fair product of skin, feel satisfied after having 91 experiment usages, and find that product is after using seven days in 108 experiments through the experiment detection, the fair nature changes the skin result to sign now, the result is rather good.

Chen owned a bio-chemical Ph.D. degree of British Oxford university the Yao breadth the professor said, the A was sour, the tartaric acid changes a skin to equalize to learn a way is currently the most familiar fair way is on the market, mainly with low Ph value acidity to decay the skin surface layer, force epidermis to shed off to reach the You Zuan AN of changing the skin however because the person's skin is rather weak, excessive usage these fair way, the serious words will cause the skin is red and swollen, ache, even have already disfigured possibility.

The breadth of Chen Yao indicates he develops to announce of transmute hormone is on planting thing He Er to receive, the He Er of its You Dun P animal receives alike can promote the cell activate as the placenta vegetable, accelerate the skin metabolism, transmute the hormone let the skin"grow up first behind peeling off" of way, since health and then nature, also can't if the animal He Er receives the danger that the sort has already infected various animal virus.

Chen the Yao breadth the professor also suggests the female friend of the love of beauty, fair change the skin result in winter the best, maintain besides and nightly also rather important, generally speaking, at 1-4:00 A.M. this time, the abruption renewal of the cell's activating speed is 800% of daytime, must match good daytime to defend to insolate a work besides, fair can show results, want to make the skin white and delicate water again bright female friend, in addition to depending on fair skin care products, defending to insolate the foundation effort can't be few.