Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori.
ABSTRACT In various insects, 20-hydroxyecdysone (20E) is indispensable for embryonic development. In eggs of the silkworm Bombyx mori, 20E has been demonstrated to be produced by two metabolic pathways: de novo synthesis from cholesterol and dephosphorylation of ovary-derived physiologically inactive ecdysteroid phosphates. In the former, ecdysone 20-hydroxylase (E20OHase) has been suggested to be a key enzyme. In the latter, it has been demonstrated that the dephosphorylation of ecdysteroid phosphates is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (EPPase). In this study, a cDNA encoding E20OHase was cloned from 3-day-old nondiapause eggs of B. mori and sequenced using PCR techniques. The protein exhibited the signature sequences characteristic of P450 enzymes, and mediated the conversion of ecdysone to 20E using the baculovirus expression system. Semi-quantitative analysis revealed that the E20OHase mRNA is expressed predominantly during gastrulation and organogenesis in nondiapause eggs, but is scarcely detected in diapause eggs whose development is arrested at the late gastrula stage. The developmental changes in the expression patterns of E20OHase and EPPase suggest that both enzyme activities are regulated at the transcription level, and both enzymes contribute cooperatively to 20E formation during embryonic development.
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ABSTRACT: We investigated here the ecdysteroid titers and the expression of six genes coding for known enzymes of the ecdysteroid biosynthesis in the testes of last instar larvae of the pest cotton leafworm, Spodoptera littoralis. We showed that the timing of the ecdysteroid profile was the same in testes and in hemolymph, with a small peak at day 2 and a large one at day 4 after ecdysis. Ecdysone and 20-hydroxyecdysone (20E) were detected in both tissues. 20E was the major ecdysteroid in testes and in hemolymph from day 4. Interestingly, the gene expression of the steroidogenetic enzymes, Neverland, and the five cytochrome P450 enzymes encoded by the Halloween genes was confirmed in the testes, and varied during the instar. However, from the data obtained so far, we cannot conclude that the measured ecdysteroids in the testes result from the activity of the genes under study. Indeed, it is suggested that the ecdysone produced centrally in the prothoracic glands, could have been transformed into 20E in the testes, where Sl-shade is well expressed.Archives of Insect Biochemistry and Physiology 09/2012; · 1.52 Impact Factor
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ABSTRACT: To examine the views and experiences of staff and users of Citizens Advice Bureau (CAB) services located in general practice, and to identify key factors perceived as contributing to the intervention's effectiveness. A qualitative study in an urban and rural primary care setting in the UK. Semi-structured, face-to-face interviews (n = 22) with primary care and practice staff, CAB advisors and 12 service users. Key positive service features reported by all groups were: the confidential, non-stigmatizing and familiar environment of a general practitioner's (GP) surgery; the ability to make appointments and experienced advisor availability and continuity. Outcomes for service users were described as financial gain, managed debt, and beneficial social and mental health impacts. Perceived staff benefits were appropriate referral and better use of GP consultation time. Welfare advice in primary care has financial benefits and was perceived by participants to offer health and other benefits to patients and staff. However, while perceptions of gain from the intervention were evident, demonstration of measurable health improvement and well-being presents challenges. Further empirical work is needed in order to explore these complex cause-effect links and the cost-effectiveness of the intervention.Public health 09/2011; 125(10):704-10. · 1.26 Impact Factor
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ABSTRACT: Steroid hormones ecdysteroids regulate varieties of developmental processes in insects. Although the ecdysteroid titer can be increased experimentally with ease, its artificial reduction, although desirable, is very difficult to achieve. Here we characterized the ecdysteroid-inactivating enzyme ecdysteroid-22-oxidase (E22O) from the entomopathogenic fungus Nomuraea rileyi and used it to develop methods for reducing ecdysteroid titer and thereby controlling insect development. K(m) and K(cat) values of the purified E22O for oxidizing ecdysone were 4.4 μM and 8.4/s, respectively, indicating that E22O can inactivate ecdysone more efficiently than other ecdysteroid inactivating enzymes characterized so far. The cloned E22O cDNA encoded a FAD-dependent oxidoreductase. Injection of recombinant E22O into the silkworm Bombyx mori interfered with larval molting and metamorphosis. In the hemolymph of E22O-injected pupae, the titer of hormonally active 20-hydroxyecdysone decreased and concomitantly large amounts of inactive 22-dehydroecdysteroids accumulated. E22O injection also prevented molting of various other insects. In the larvae of the crambid moth Haritalodes basipunctalis, E22O injection induced a diapause-like developmental arrest, which, as in normal diapause, was broken by chilling. Transient expression of the E22O gene by in vivo lipofection effectively decreased the 20-hydroxyecdysone titer and blocked molting in B. mori. Transgenic expression of E22O in Drosophila melanogaster caused embryonic morphological defects, phenotypes of which were very similar to those of the ecdysteroid synthesis deficient mutants. Thus, as the first available simple but versatile tool for reducing the internal ecdysteroid titer, E22O could find use in controlling a broad range of ecdysteroid-associated developmental and physiological phenomena.Journal of Biological Chemistry 03/2012; 287(20):16488-98. · 4.65 Impact Factor