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Phytoecdysteroids, which are structurally similar or identical to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. To study the mechanism of action of phytoecdysteroids in mammalian tissue, an in vitro cellular assay of protein synthesis was developed. In C2C12 murine myotubes and human primary myotubes, phytoecdysteroids increased protein synthesis by up to 20%. In vivo, ecdysteroids increased rat grip strength. Ecdysteroid-containing plant extracts produced similar results. The effect was inhibited by a phosphoinositide kinase-3 inhibitor, which suggests a PI3K-mediated mechanism.

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(D.M.R., N.I., T.J.C.)l h e effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H,]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled interna1 standards.[l3C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [ZH,lIAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. l h e presente of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.Since the 1940s (Thimann, 1974), exogenous auxins have been used frequently as experimental tools for studying their effects in developing structures (Schiavone and Cooke, 1987); for screening for mutants impaired in the uptake, metabolism, and physiology of endogenous auxins (Timpte et al., 1994); and even to find potential IAA receptors (Jones and Venis, 1989; LoSchiavo et al., 1991). One important use for the application of exogenous auxin is as a growth regulator for maintaining plant-cell-and tissue-

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The genetic advantages t o the use o f Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods t o determine amounts and examine the metabolism of indolic compounds in Arabidopsis. lsolation and identification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAAI) was carried out under mild conditions, thus proving i t s natural occurrence. We describe here the synthesis of 13Cl-labeled I A N and i t s utility i n the gas chromatography-mass spectrometry quantification o f endogenous IAN levels. We also quantified the nonenzymatic conversion o f I A N t o IAA under conditions used t o hydrolyze IAA conjugates. 13C1-Labeled I A N was used t o assess the contribution of I A N t o measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known t o be present i n Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.The study of plant hormone biogenesis and metabolism has been enhanced greatly by the application of genetic techniques, especially the use of metabolic mutants (Normanly et al., 1995). Various plants have been used for such biochemical genetic studies and these include maize, pea, tobacco, and Lemna gibba. The use of Arabidopsis for such studies has significant advantages because of the simplicity of its genome, short life cycle, and the existence of an impressive number of mutant plants and information concerning specific genes. For studies of auxin metabolism, however, the advantages associated with Arabidopsis pre-