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Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis

Department of Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 4000 Roskilde, Denmark.
Molecular and Cellular Endocrinology (Impact Factor: 4.24). 04/2006; 247(1-2):166-74. DOI: 10.1016/j.mce.2005.12.053
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ABSTRACT The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active insect molting hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.

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    • "E20M) is the insect cytochrome P450-dependent steroid hydroxylase responsible for the conversion of the arthropod molting hormone ecdysone (E) to its more active metabolite, namely, 20-hydroxyecdysone (20E) [1] [2]. The nature, regulation, and molecular biology of E20M were elucidated predominantly employing the tobacco hornworm, Manduca sexta, as the model [3] [4] [5] [6] [7] [8]. Indeed, the developmental impact of E20M was made evident in midgut tissue of the M. sexta fifth larval instar where E20M activity increases 50-fold between days four and five of the stadium and this increase is inextricably tied to the onset of wandering stage behavior [1] [8] [9]. "
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    ABSTRACT: The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone), and five anthraquinones that were synthesized from quinizarin, viz., 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect model Manduca sexta (tobacco hornworm). This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form. M. sexta fifth larval instar midgut homogenates were incubated with increasing concentrations (10-8 to 10-3 M) of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibited I50’s of about 4 x 10-6 to 6 x 10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4 –anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.
    International Journal of Zoology 10/2014; 2014:8. DOI:10.1155/2014/261512
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    • "This suggests that other tissues may have roles in ecdysteroid biosynthesis. According to previous studies, Malphigian tubules may release 20E into the hemolymph, whereas the midgut accumulates polar ecdysteroid metabolites prior to their excretion (Feyereisen et al. 1978, 1980; Rewitz et al. 2006b), which indicates that the Malpighian tubules may function not only in the excretion of 20E but also in maintaining the hemolymph 20E titer that is elicited during molting to the pupa. Moreover, recent work showed that 20E was involved in the differentiation of stem cells from the midgut of the caterpillar S. littoralis (Smagghe et al. 2005). "
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    ABSTRACT: Abstract 20-Hydroxyecdyone, an active form of ecdysteroid, is the key hormone in insect growth and development. Halloween genes encode ecdysteroidogenic enzymes, including cytochrome P450 monooxygenase. CYP307A1 (spook) is accepted as an enzyme acting in the so-called 'black box' that includes a series of hypothetical and unproven reactions that finally result in the oxidation of 7-dehydrocholesterol to diketol. In this study, the Holcocerus hippophaecolus Hua (Lepidoptera: Cossidae) CYP307A1 (HhSpo) gene was identified and characterized. The obtained cDNA sequence was 2084 base pairs with an open reading frame of 537 animo acids, in which existed conserved motifs of CYP450 enzymes. The transcript profiles of HhSpo were analyzed in various tissues of final instar larvae. The highest expression was observed in the prothoracic gland, while expression level was low but significant in other tissues. These results suggest that the sequence character and expression profile of HhSpo were well conserved and provided the basic information for its functional analysis.
    Journal of Insect Science 06/2013; 13:56. DOI:10.1673/031.013.5601 · 0.92 Impact Factor
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    • "). Unlike the other Halloween genes, transcription of this gene 69 mainly takes place in peripheral tissues, such as midgut, epidermis, 70 fat body and Malpighian tubules of immature insect stages (Petryk 71 et al., 2003; Rewitz et al., 2006a,b). The description/prediction of 72 the Halloween genes in other insect species is mostly based on 73 sequence similarity (Christiaens et al., 2010; Iga and Smagghe, 74 2010; Yamazaki et al., 2011). "
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    ABSTRACT: A major breakthrough in elucidating the ecdysteroid biosynthetic pathway in insects was realized with the molecular identification and further functional characterization of the 'Halloween' genes. These genes were found to encode cytochrome P450 enzymes catalysing the final steps of ecdysteroid biosynthesis in the dipteran, Drosophila melanogaster, and in the Lepidoptera, Manduca sexta and Bombyx mori. A recent report focused on the identification of Halloween orthologs in the desert locust, Schistocerca gregaria, a member of the hemimetabolous insect order of the Orthoptera. In the present study, an additional Halloween gene Shade, is identified in the desert locust. In Diptera and Lepidoptera, this gene encodes a 20-hydroxylase, catalysing the conversion of ecdysone (E) to 20-hydroxyecdysone (20E). However, this enzymatic function has previously been suggested for CYP6H1 in another locust species, the migratory locust, Locusta migratoria. Using q-RT-PCR, the spatial and temporal transcript profiles of S. gregaria orthologs for Shade as well as CYP6H1 were analysed in last larval stage desert locusts. An RNA interference (RNAi)-based approach was employed to study whether these genes could possibly encode a functional 20-hydroxylase in the desert locust.
    Journal of insect physiology 03/2012; 58(7):890-6. DOI:10.1016/j.jinsphys.2012.03.013 · 2.50 Impact Factor
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