Ecdysteroid preparation

To verify in vitro results, we have also developed an in vivo assay for L. dispar PTTH (52, Thyagaraja et al, in preparation). This assay is comparable to larval assays develop for Af. sexta (56 and B. mori (58) and uses last instar female larvae. Neck ligation prior to day 7, the day when a small pre-peak in hemolymph ecdysteroid titer appears, blocks further development. Injection of PTTH-containing extracts posterior to the ligation, including post-embryonic egg extract, reinitiates development and subsequent pupation. Attempts to develop an in vivo assay comparable to the pupal-adult B. mori assay (5B were unsuccessful, since brain removal as close as 15 min post-pupal ecdysis failed to block L. dispar female adult development (Thyagaraja et a/., unpublished results). 

M. domestica oostatic hormone seems to Inhibit the release or synthesis of egg development neurosecretory hormone (EDNH) (12), but in mosquitoes the hormone seems to act directly on the ovary (6). R, prolixus oostatic hormone, which has been partially purified (13), is a small peptide of M, 1,411 as determined on Sephadex G-50. Kelly et al. (5) prepared a crude extract of oostatic hormone from M, domestica, injected it into the autogenous mosquito Aedes atropalpus, and demonstrated inhibition of both egg development and ecdysteroid biosynthesis. They suggested that oostatic hormone functions at a point subsequent to release of EDNH. They were unable, however, to exclude the possibility that release of EDNH, itself, was inhibited. 

Ecdysteroids All ecdysteroids used in this work were a kind gift of Prof. Dr. Jan Koolmann, University of Marburg. They were ecdysone, 20-hydroxyecdysone, makisterone and muristerone for the growth experiments, as well as radiolabeled ponasterone A for the binding assay. Preparation of stock solutions, sterilization and addition to media was exactly the same as for brassinosteroids. 

These experiments with enriched ecdysteroid receptor preparations demonstrate the potential of some brassinosteroids to interact with ecdysteroids at the target site of the hormone. However, this does not exclude other sites of interference with ecdysteroids. Theoretically, brassinosteroids may also affect the hormone system at the site of transportation in the haemolymph, at the sites of enzymatic conversion of ecdysteroids or at the excretory system. 

There are several reports that cHH can inhibit the ecdysteroid synthesis by YO in vitro but that MIH cannot act as a cHH by increasing glucose concentration (see for example [108]). It was probably this multifunctionality of cHH that led to it being mistaken for MIH in certain instances. Why should cHH duplicate the role of another hormone during the moult cycle The definitive answer to this question is still not known but specific receptors for cHH (along with MIH-specific receptors) have been demonstrated, by classical membrane binding studies, to be present on the YOs of intermoult brachyuran crabs [84]. Unlike cHH, radiolabelled MIH binds only to membrane preparations of YOs and not... 

Sterols are involved in the stabilization of cell membranes (E 2.2). Ergosterol is used in the technical preparation of vitamin Dg (D 6.4.9). Ecdysone and ecdy-sterone are molting hormones in insects and crustaceans (E 3.1). Similar compounds (ecdysteroids), e.g., ponasterone and cyasterone, which are widespread in higher plants, deter feeding insects (E 5.5.3). The hormone activity of certain ecdyste-roids in insects is up to 20 times that of ecdysone. Certain Drosophila species are... 

Lamparczyk (1992) provided extensive coverage of the chromatographic analyses of steroids and updated information in Touchstone (1986). The book includes information on steroid nomenclature, chromatographic methods used in steroid analyses, and sample preparation. Although not restricted to TLC, the book provides extensive information on all aspects of TLC related to androgens, bile acids, ecdysteroids, estrogens, pregnanes, and corticoids. 

Wilson (1992) described the use of various boronic acids for the derivatiza-tion and separation of ecdysteroids by normal- and reversed-phase TLC. The derivatives were prepared from butyl-, phenyl-, and aminophenylboronic acids by over-spotting a methanolic solution of the reagent on to the applied ecdysteroids at the origin of the TLC plate. Butylboronic acid was also used as a mobile-phase additive to modify chromatographic behavior. A 20,22-diol group was an essential structural requirement for the formation of a cyclic boronate of an ecdysteroid. These procedures allowed for the selective separations of such compounds as ponasterone A and 2-deoxyecdysone. Pis and Harmatha (1992) also described the use of phenylboronic acid as a versatile derivatization agent for TLC studies of ecysteroids. 

Fell (1996) has provided useful information on analytical techniques and approaches for the separation of ecdysteroids by TLC. He noted that ecdysteroid separation can be achieved on silica gel TLC plates, HPTLC plates, RP-TLC and RP-HPTLC plates. Plates with a fluorescent indicator (F254) allow for ecdysteroid detection visually by UVC (fluorescence quenching). Paraffin-coated plates can be used (by developing plates in dichloromethane containing 5 to 7.5% paraffin oil) to improve the separation and recovery of ecdysteroids. For details on the preparation of these plates, see Fell (1996). For a practical approach to the analysis of ecdysteroids by TLC including methods of sample preparation, choice of chromatographic systems (i.e., combination of plates and solvent systems) and methods of detection, see Fell (1996). 

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