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Increasing evidence exist that multiple G proteins mediate the effects of gonadotropin-releasing hormone (GnRH) on the synthesis and release of pituitary gonadotropins. In the present study, we have expressed the rat GnRH receptor (GnRH-R) in insect cells, by infection with a recombinant baculovirus. Under the conditions used, insect cells expressed, 48 h post-infection, a maximum of 7800 +/- 650 receptors/cell which bound GnRH agonist [D-Trp6]GnRH with a Kd = 0.52 +/- 0.06 nM indicating characteristics similar to those of the natural receptor. No binding was observed in non-infected cells or cells infected with wild-type baculovirus. In presence of GnRH, GnRH-R expressing cells elicited a time- and dose-dependent production of inositol trisphosphate, with a maximum level reached within 30 min and an EC50 = 5 nM. These recombinant insect cells also produced cAMP in response to GnRH. However, in contrast to other heterologous systems, or rat pituitary gonadotropes wherein GnRH induced a weak and delayed elevation of cAMP, in insect cells the rise of cAMP was comparatively rapid, attaining a maximum level after 2 h, and the EC50 was 5 nM. Finally, a clear activation of adenylyl cyclase (AC) in response to GnRH was shown for the first time by measuring the conversion of [alpha-32P]ATP into labeled cAMP, using membrane preparations from GnRH-R expressing insect cells. These data demonstrate that rat GnRH-R has the potential for dual coupling to both phosphoinositidase C and AC and suggest a major influence of the host cell for this coupling and/or its expression, probably in relation with the G protein repertoire and preference. This notion could be extended to several target cells other than pituitary gonadotropes that normally express the GnRH-R in mammals, including hippocampal, Leydig, granulosa, placental and GnRH-secreting hypothalamic cells.

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Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common alpha- and a specific beta-subunit. A recombinant baculovirus containing a cDNA encoding the alpha-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7-10 mg immunoreactive alpha-glycopolypeptide/6 x 10(8) cells, of which 65-6% was able to associate with native LH beta and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant alpha-subunit was produced as two differently glycosylated forms; an M(r) 23 000 form which contained exclusively N-linked carbohydrate units and another of M(r) 25 000 which appeared to contain additional 0-linked carbohydrate. Data demonstrated that the alpha-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated alpha-subunit is known to be capable of associating with the beta-subunit.

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A chimeric Xdh gene was constructed in vitro, by recombining DNA sequences from the Dipterans Drosophila melanogaster and Calliphora vicina. The ry506 strain, an eye-colour mutant of Drosophila that is deficient for Xdh, was genetically transformed with the recombinant gene. Transformed flies with ry+ eye phenotype and increased resistance to purine were obtained, showing that the chimeric XDH is physiologically active in Drosophila. XDH activity was detected in crude extracts from transformed flies, yet at lower levels than in wild-type controls. The amounts of Xdh transcripts in the transformants were found to be 8 to 16% of the amount of wild-type ry mRNA, suggesting that Calliphora Xdh sequences may be relatively inefficient for mRNA production in Drosophila, or may produce unstable mRNA.

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Farnesylacetone (C18 H30 0) is a male hormone extracted from the androgenic gland of crab, Carcinus maenas. Appropriate enzymatic assays, as well as spectrophotometric studies, indicate that micromolar concentrations of farnesylacetone interact with the electron transport pathway of rat liver mitochondria. By the use of artificial electron donors and electron acceptors, it is shown that farnesylacetone immediately inhibits the electron transfer within complex I (NADH ubiquinone reductase activity) and complex II (succinate ubiquinone reductase activity). It is proposed that farneylacetone could interact with these two complexes of the respiratory chain at the level of the iron-sulfur centers implicated in the dehydrogenase activities. These observations are compared with the results obtained with terpenic molecules which interact with mitochondrial respiration.

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In vitro enzymatic assays have shown that an enzyme with typical xanthine dehydrogenase (XDH) activities and electrophoretic mobility slightly different from that of Drosophila XDH is present in Calliphora tissues. A Calliphora genomic sequence has been isolated by low-stringency hybridization to the Drosophila rosy gene (XDH), and partially sequenced. This sequence has been shown to be unique, polymorphic, and it maps on chromosome I. Sequence comparisons provide compelling evidence that it belongs to the XDH gene of Calliphora. Interspecies transformation experiments, aimed at investigating functional as well as structural divergence of the XDH genes of Calliphora and Drosophila, are now possible.

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A library of Calliphora vicina genomic DNA was constructed in the lambdaEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A) RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A) RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5-5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1beta gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.

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The temperature-sensitive 1(3)ecd-1ts mutation (A. Garen, L. Kauvar, and J.A. Lepesant (1977). Proc. Natl. Acad. Sci USA 74, 5099-5103.) has been used in several laboratories to obtain Drosophila larvae deprived of moulting hormone. The development of mutants and controls during the third larval instar at permissive (20 degrees C) and restrictive temperatures (29 degrees C) was compared. Pupariation was inhibited when larvae were shifted to the restrictive temperature immediately at the second moult. The permanent larvae obtained remained active, did not leave the food, and reached a maximum weight superior to the weight of controls. Ecdysteroids were studied during the third larval instar by HPLC analysis and radioimmunoassays. A careful synchronization of the larvae at the second moult enabled the confirmation that at least one ecdysteroid peak occurs during the third larval instar, prior to the wandering stage in controls (20 or 29 degrees C). Ecdysone was then the predominant moulting hormone, whereas 20-hydroxyecdysone was the main ecdysteroid at the time of pupariation. Low levels of ecdysteroid were measured in mutant larvae shifted to 29 degrees C immediately at the second moult but larvae completely deprived of immunoreactive material were never observed. Nearly normal levels of ecdysteroids appeared at 27.5 degrees C. Feeding ecd-1 larvae maintained at restrictive temperature on 20-hydroxyecdysone-yeast mixture for 16 hr triggered abortive pupariation. Ecdysteroid levels were measured after the return of the larvae to the standard medium; normal levels were restored 24 hr later. The mutant ecd-1 appears to present interesting opportunities for the detailed study of the hormonal induction of a developmental process during the third larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)

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Poly A (+)mRNA synthesis was analyzed during the nymphal stage and the diapause in the wing discs of the Lepidopteran Pieris brassicae. The main events of differentiation, i.e. scale formation, adult cuticle elaboration and pigment deposition, occurred during the period studied. We only detected one phase of synthesis for poly A (+)mRNA molecules, 48-72 hours after the nymphal moult. This synthesis was found to be related to that of late proteins at 120-140 hours. Examination of mRNA metabolism in discs treated with cordycepin (3' dA) showed a decline in mRNA stability. This decline corresponded to a turn-over phase followed by a period of stabilization which preceded mRNA translation. In diapausing animals, poly A (+)mRNA metabolism was unexpectedly high, and many messengers were synthesized and rapidly destroyed. These messengers were found to be slightly heavier than the mRNAs produced during normal development, suggesting a blocking at some step in their maturation. We developed a mathematical model for mRNA metabolism which enabled us to calculate the effects of synthesis and degradation on the quantity of mRNAs, from the poly A (+)mRNA concentration and the turnover time in the mRNA pool. In addition determination phases associated with embryological events and terminal differentiation are clearly distinguished. This feature offers opportunities to investigate the commitment events which take place during the end of the larval stage and the beginning of the nymphal stage.

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This work describes a model for simulation of rRNA and polyA(+)mRNA evolution during a developmental process - the wing disc differentiation of an Insect, Pieris Brassicae. The model was constructed in two sections: --a logical section built around a logical network of genetic regulations for RNA synthesis and degradation. We postulated the existence of clock pulses, which act as specific stages of development on parameters determining the accumulation and disappearance of the different RNA classes. A simple model was thus able to account for the complex variations of the studied macromolecules during the differentiation phase. --an analogical section, to account for and calculate the accumulation of terminal gene products. This part of the model was based on essential features of the automata theory. In addition, the relations between clock pulses, biological rhythms and their alteration during diapause are discussed. Diapause and emergence from diapause were included in the above model for RNA metabolism. This model enabled us to compute the time at which particular events take place and the stages of synthesis of the main RNA classes. We evaluated the synthesis and degradation rates of these different classes of molecules. These rates are closely related to those reported by other authors in several differentiation systems. Our model seems applicable to any developmental process in which RNA synthesis exhibits noticeable variations. Although such an approach has seldom been used to interpret macromolecular expressions, its application in the present case allowed us to obtain by a simple computation, complex information on the genetic and biochemical features of RNA molecule regulation.

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Regeneration was induced in the imaginal discs in situ following lesions caused by heat-sensitive cell-lethal mutations. A clonal analysis of this event demonstrated that the subsequent delay in pupariation was correlated with the amount of extra growth that occurred during the regeneration. Pupariation of heat-treated gynandromorphs bearing the mutations was also retarded, and the duration of larval development increased with greater amounts of mutant tissue, it was therefore correlated with the extent of the lesions in the imaginal discs. Elimination of entire imaginal discs, or the presence of very small amounts of lethal tissue, did not result in prolonged larval life.

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Larvae of the fly Calliphora erythrocephala (Meigen) were deprived surgically of their ring glands at an age prior to the appearance of ecdysone in the blood, and then injected with ecdysone. They contracted into the typical barrel-shaped puparium, before the onset of tanning. This proved that ecdysone controls the puparium contraction as well as tanning.

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