Endogenous inhibitor of ecdysone synthesis in crabs

Summary Attempts to isolate the molt-inhibiting hormone (MIH) of crustaceans from crab eyestalks (ES) resulted in the characterization of xanthurenic acid as an inhibitor of ecdysone biosynthesis in the cultured Y-organ-complex (YOC) homogenate. It was also found that 3-hydroxyl-l-kynurenine present in the ES is transformed into xanthurenic acid in the YOC and body fluid. Its mode of inhibitory action in ecdysone biosynthesis is probably inactivation of cytochrome P-450.Acknowledgments. We thank Prof. M. Oka (Nagasaki University) and Dr J. Cappuzo (Oceanographic Inst.) for information on Y-organ and molting stages, Dr J. Termini and Mr J. Cesarelli (Columbia University) for organ excision of blue crabs and experiments in the early stage of this study, the Japan Sea Farming Association for aqua culture of crabs, Prof. Y. Umebachi (Kanazawa University) for a gift of 3-OH-l-Kyn, and Mr J. Rudloe (Gulf Specimen Co.) for collection and shipping of some selected crabs. This study was partially supported by NIH grant AI 10 187 (to K. N., at Columbia University).     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone in representative insects

The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

Abnormal kynurenine pathway of tryptophan catabolism in cardiovascular diseases

Kynurenine pathway (KP) is the primary path of tryptophan (Trp) catabolism in most mammalian cells. The KP generates several bioactive catabolites, such as kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), xanthurenic acid (XA), and 3-hydroxyanthranilic acid (3-HAA). Increased catabolite concentrations in serum are associated with several cardiovascular diseases (CVD), including heart disease, atherosclerosis, and endothelial dysfunction, as well as their risk factors, including hypertension, diabetes, obesity, and aging. The first catabolic step in KP is primarily controlled by indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). Following this first step, the KP has two major branches, one branch is mediated by kynurenine 3-monooxygenase (KMO) and kynureninase (KYNU) and is responsible for the formation of 3-HK, 3-HAA, and quinolinic acid (QA); and another branch is controlled by kynurenine amino-transferase (KAT), which generates KA. Uncontrolled Trp catabolism has been demonstrated in distinct CVD, thus, understanding the underlying mechanisms by which regulates KP enzyme expression and activity is paramount. This review highlights the recent advances on the effect of KP enzyme expression and activity in different tissues on the pathological mechanisms of specific CVD, KP is an inflammatory sensor and modulator in the cardiovascular system, and KP catabolites act as the potential biomarkers for CVD initiation and progression. Moreover, the biochemical features of critical KP enzymes and principles of enzyme inhibitor development are briefly summarized, as well as the therapeutic potential of KP enzyme inhibitors against CVD is briefly discussed.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3β-reductase mediated conversion to ecdysone in representative insects

Summary The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

External factors and ecdysone release inCalliphora erythrocephala

Summary Ecdysone level is measured in the haemolymph of normal larvae and of larvae submitted to external stimulation. External stimulation delays the release of ecdysone and the formation of puparium. The adaptative value of the mechanism which connects the ecdysone titre to external stimulation is discussed.     

RH-5849, a nonsteroidal ecdysone agonist,does not mimic makisterone-A inDysdercus koenigii

The response of final instar nymphs ofDysdercus koenigii to topical application of the non-steroidal ecdysone agonist, RH-5849, was dose dependent. The candidate compound produced mortality even at moderate doses, but precocious adult development was not observed. Similar results were obtained after oral administration or injection. Conversely, injections of makisterone-A (the principal moulting hormone ofDysdercus) into 5th instar nymphs resulted in precocious adult development within 4 days. We conclude that RH-5849 does not mimic makisterone-A, as is the case with ecdysone, and that toxicity is mediated instead through non-endocrine targets in this insect species.     

Regulatory steps in sex pheromone biosynthesis inMamestra brassicae L. (Lepidoptera: Noctuidae)

Summary The de novo biosynthesis of (Z)-11-hexadecenyl acetate, the most abundant pheromone component inM. brassicae, starting from acetate via palmitic acid, requires the presence of a pheromone-biosynthesis-activating neurohormone. Moreover, the conversion of palmitic acid to (Z)-11-hexadecenyl acetate is strongly dependent on the presence of the neurohormone. However, no significant dependence was found for the conversion of (Z)-11-hexadecenoic acid to (Z)-11-hexadecenyl acetate. This indicates that the neurohormonal control of pheromone biosynthesis inM. brassicae occurs at the level of palmitic acid.     

Variations des taux d'ecdystéroïdes au cours du développement deBombyx mori; rapport entre ces variations et les phases de croissance et de morphogenèse

Summary During the development ofBombyx mori (monovoltin race) ecdysteroid levels were determined in oocytes, eggs, and haemolymph of larvae, and in the haemolymph of pupae. In haemolymph, the only RIA reactive materials are ecdysone and ecdysterone. In oocytes and eggs, other ecdysteroids are also detected. During larval instars, the ecdysteroid levels are low whereas they are very high during morphogenetic periods. During embryonic diapause, the ecdysone titer decreases during the cessation of morphogenesis.     

The generality of the nitroso-glyoxylate reaction: Conversion ofp-nitronitrosobenzene to the hydroxamic acid

Summary The reaction of glyoxylic acid withp-nitronitrosobenzene (1b) in dilute aqueous solution gave the hydroxamic acid (2b) as the major detectable product. The significance of this observation with respect to the title reaction is discussed.This study was supported by grant No. CA 32395 from the National Cancer Institute, and by Research Career Development Award ES 00120 from the National Institute of Environmental Health Sciences, DHHS.     

Effects of the pyrimidine-containing cytochrome P-450 inhibitor,fenarimol, on the formation of 20-OH ecdysone in flies

Summary The effect of fenarimol, a pyrimidine-containing cytochrome P-450 inhibitor, was tested in vitro on brain-ring gland complexes ofCalliphora vicina (Dipt., Calliphoridae), and on microsomes prepared from the fat body of 0-h wandering stage larvae ofNeobellieria bullata (Dipt., Sacrophagidae). Fenarimol had no influence on the formation of ecdysone, but it was an effective inhibitor of cytochrome P-450-dependent ecdysone 20-monooxygenase.     

A peculiar fatty acid, (Z,Z)-9,12,17-octadecatrienoic acid,identified in the phospholipids of the pea aphid,Acyrthosiphon pisum (Harris) (homoptera: aphididae)

A peculiar fatty acid previously detected in the phospholipids of the pea aphid,Acyrthosiphon pisum, is identified as (Z,Z)-9,12,17-octadecatrienoic acid. It is the first report of this compound in the literature. Comparison of fatty acid profiles of phospholipids between normal and aposymbiotic pea aphids shows that aphid symbionts are not responsible for the biosynthesis of this unusual fatty acid.     

Occurrence of ecdysone in the blood of the chelicerate arthropod,Limulus polyphemus

Summary A molt promoting substance, assumed to be ecdysone, was discovered inLimulus haemolymph. Preliminary bioassay suggests a titre of 9 ng ecdysone per ml of haemolymph.Supported by the University of Minnesota Graduate School, NSF Grant No. GB 16607 and USPHS Grant No. HO 07336.     

Critical period and ecdysone titers in the pupae ofPieris brassicae

Summary The ecdysone titer in the haemolymph of pupae of Pieris brassicae shows a sharp peak (1.90 g/ml) on days 5 and 6 of the pupal stage. Isolated abdomens were able to form adult abdomens at 72 h, this being 2 or 3 days before maximum ecdysone levels and at a time when the concentration of hormone in the abdominal haemolymph was only 0.23 g/ml.     

Interaction ofRhizobium loti strain and host on the biosynthesis of unusual amino acids in leguminous plants

Summary The effect of differentRhizobium loti strains on the biosynthesis of 2,3-diamino-butanoic acid and 2,4-diamino-3-methyl-butanoic acid in root nodules ofLotus tenuis, Anthyllis vulneraria andLupinus densiflorus has been investigated. Results suggest that biosynthesis isRhizobium strain dependent, that the bacteroid is the site of synthesis of the compounds and that their biosynthesis is confined to the symbiosis.     

Ecdysteroid hormone action

Several reviews devoted to various aspects of ecdysone research have been published during the last few years. Therefore, this article concentrates mainly on the considerable progress in ecdysone research observed recently, and will cover the results obtained during the last 2 years. The main emphasis is put on the molecular mode of ecdysteroid receptor-mediated hormone action. Two examples of interaction with other hormonal signalling pathways are described, namely crosstalk with juvenile hormone and insulin. Some selected, recently investigated examples of the multitude of hormonal responses are described. Finally, ecological aspects and some practical applications are discussed.     

Direct observation of enzyme substrate complexes by stopped-flow fluorescence: Mathematical analyses

Summary The fluorescence changes which occur upon the interaction of enzyme and substrate under stopped-flow conditions can provide a sensitive means to directly observe ES complexes. The interconversion of the intermediates during catalysis causes changes in fluorescence, signaling directly their existence, and allowing their quantitation. We have studied extensively and approach which measures radiationless energy transfer (RET) between enzyme tryptophanyl residues and a fluorescent peptide or ester substrate. Our studies of a number of proteolytic enzymes have validated the approach, which is sensitive and applicable to a variety of enzymes under a wide range of experimental conditions, including subzero temperatures. Direct excitation of fluorescent substrates can also be used to observe ES complex formation and breakdown and is complementary to the RET approach. Here we review both the RET and direct excitation kinetic approaches, with particular emphasis on the mathematical foundations we have developed which are critical to the successful interpretation of these or any other spectroscopic approach which yields a signal that is unique to ES complex.Acknowledgment. This work was supported by Grants-in-Aid GM-24967 and 24968 from the National Institute of Health of the Department of Health, Education and Welfare to Harvard Medical School. The continued advice and support of Dr B.L. Vallee is gratefully acknowledged, as is the help of Dr L. Bethune on theoretical aspects of the steady-state assumption. The excellent technical assistance of P. Maiorana is acknowledged. The abbreviations used are: RET; radiationless energy transfer, Dns, or dansyl; 5-dimethylaminonaphthalene-1-sulfonyl; DED; dansylethylenediamine, Mes; 2-(N-morpholino)ethane sulfonic acid, OMe; methoxide, mansyl; 6-(N-methylanilino)-naphthalene-2-sulfonyl.     

Haemolymph ecdysteroids level following the injection of ecdysone or ecdysterone; its relation with tegument and midgut response inAeshna cyanea (Insecta,Odonata)

Summary The haemolymph ecdysteroid level after injection of ecdysone or ecdysterone inAeshna cyanea larvae has been determined by a radioimmunoassay method. The rate of excretion appears to be dependent on both the ecdysteroid injected and the time of injection. In case of ecdysone injection, the secretion of the epidermis cuticle and the differentiation of the imaginal midgut epithelium occur when the ecdysteroid level remains low for many days.     

The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell

Control of mammalian gene promoters by the bacterial LacI repressor provides reversible regulation and dose-response levels of derepressed expression by the lactose analog isopropyl thiogalactose (IPTG). Here, we show that insertion of LacI-binding sites in the ubiquitous β-actin promoter confers a strong and dose-dependent IPTG-regulatable expression of transiently transfected reporter genes in mouse embryonic stem (ES) cells expressing LacI. We established ES cell lines stably expressing reporter genes under inducible control and found a five- to tenfold IPTG induction of transgene expression. The kinetics of induction is rapid and stable, and can be rapidly reversed after IPTG removal. Importantly, this regulatable expression was maintained throughout the differentiation process of ES cells, and observed in individual differentiated cardiomyocyte-like cells and neuronal-like cells. This reversible system is the first to function from undifferentiated to individual welldifferentiated ES cells, providing a very useful tool to understand molecular mechanisms underlying ES cell self-renewal, commitment and differentiation.Received 17 March 2005; received after revision 19 April 2005; accepted 25 April 2005