Radioimmunoassay of arthropod moulting hormone: ß‐ecdysone antibodies production and <sup>125</sup>i‐iodinated tracer preparation

Porcheron, P.; Foucrier, Jean; Gros, C; Pradelles, P.; Cassier, Pierre; Dray, F.

doi:10.1016/0014-5793(76)81027-7

Cited by 58 publications

(21 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ecdysteroid levels were quantified by ELISA following the procedure described by Porcheron et al (Porcheron et al, 1976), and adapted by Romañá et al (Romañá et al, 1995). 20E (Sigma) and 20E-acetylcholinesterase (Cayman Chemical) were used as the standard and enzymatic tracer, respectively.…”

Section: Ecdysteroid Titers and Weight Quantificationsmentioning

confidence: 99%

“…Absorbance was read at 450 nm using a Multiscan Plus II Spectrophotometer (Labsystems). The ecdysteroid antiserum has the same affinity for ecdysone and 20E (Porcheron et al, 1976), but because the standard curve was obtained with the latter compound, results are expressed as 20E equivalents. For sample preparation, 15 staged larvae were weighed and preserved in 600 μl of methanol.…”

Section: Ecdysteroid Titers and Weight Quantificationsmentioning

confidence: 99%

See 1 more Smart Citation

Smt3 is required forDrosophila melanogastermetamorphosis

et al. 2008

View full text Add to dashboard Cite

Sumoylation, the covalent attachment of the small ubiquitin-related modifier SUMO to target proteins, regulates different cellular processes, although its role in the control of development remains unclear. We studied the role of sumoylation during Drosophila development by using RNAi to reduce smt3 mRNA levels in specific tissues. smt3 knockdown in the prothoracic gland, which controls key developmental processes through the synthesis and release of ecdysteroids, caused a 4-fold prolongation of larval life and completely blocked the transition from larval to pupal stages. The reduced ecdysteroid titer of smt3 knockdown compared with wild-type larvae explains this phenotype. In fact, after dietary administration of exogenous 20-hydroxyecdysone, knockdown larvae formed pupal cases. The phenotype is not due to massive cell death or degeneration of the prothoracic glands at the time when puparium formation should occur. Knockdown cells show alterations in expression levels and/or the subcellular localisation of enzymes and transcription factors involved in the regulation of ecdysteroid synthesis. In addition, they present reduced intracellular channels and a reduced content of lipid droplets and cholesterol, which could explain the deficit in steroidogenesis. In summary, our study indicates that Smt3 is required for the ecdysteroid synthesis pathway at the time of puparium formation.

show abstract

Section: Ecdysteroid Titers and Weight Quantificationsmentioning

confidence: 99%

Section: Ecdysteroid Titers and Weight Quantificationsmentioning

confidence: 99%

Smt3 is required forDrosophila melanogastermetamorphosis

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Absorbance was read at 450 nm using a Multiscan Plus II Spectrophotometer (Labsystems). The antiserum has the same affinity for ecdysone as for 20E (26), but because the standard curve was obtained with the latter compound, the results are expressed as 20E equivalents. For sample preparation, 15 to 20 staged larvae and pupae were weighed and preserved in 600 l of methanol.…”

Section: Methodsmentioning

confidence: 99%

“…Each harvest included experimental and control genotypes in order to ensure an objective developmental age. Ecdysteroid levels were quantified by enzyme-linked immunosorbent assay (ELISA), following the procedure previously described (26) and further adapted (27). 20E (Sigma) and 20E-acetylcholinesterase (Cayman Chemical) were used as the standard and enzymatic tracer, respectively.…”

Section: Methodsmentioning

confidence: 99%

Genes of the Ecdysone Biosynthesis Pathway Are Regulated by the dATAC Histone Acetyltransferase Complex in Drosophila

Pankotai

Popescu

Grau

et al. 2010

Molecular and Cellular Biology

View full text Add to dashboard Cite

Uncovering mechanisms that regulate ecdysone production is an important step toward understanding the regulation of insect metamorphosis and processes in steroid-related pathologies. We report here the transcriptome analysis of Drosophila melanogaster dAda2a and dAda3 mutants, in which subunits of the ATAC acetyltransferase complex are affected. In agreement with the fact that these mutations lead to lethality at the start of metamorphosis, both the ecdysone levels and the ecdysone receptor binding to polytene chromosomes are reduced in these flies. The cytochrome genes (spookier, phantom, disembodied, and shadow) involved in steroid conversion in the ring gland are downregulated, while the gene shade, which is involved in converting ecdysone into its active form in the periphery, is upregulated in these dATAC subunit mutants. Moreover, driven expression of dAda3 at the site of ecdysone synthesis partially rescues dAda3 mutants. Mutants of dAda2b, a subunit of the dSAGA histone acetyltransferase complex, do not share phenotype characteristics and RNA profile alterations with dAda2a mutants, indicating that the ecdysone biosynthesis genes are regulated by dATAC, but not by dSAGA. Thus, we provide one of the first examples of the coordinated regulation of a functionally linked set of genes by the metazoan-specific ATAC complex.

show abstract

“…Where necessary, background values (blank injection) have been subtracted. Two further antisera were used for dose-response experiments: the DHS I-15 antiserum (13), a gift of J. D. O'Connor to H. H. Rees (used at a dilution of 1:500) and an antiserum (MAS 7) generated in mice by using as antigen 20-hydroxyecdysone conjugated through C6 to bovine serum albumin (Sigma) (14,15). The resulting antiserum (pooled from five mice) was used at a dilution of 1:550 and had a Kd for ecdysone of 4.9 x 10-10 M; the affinity for 20-hydroxyecdysone was approximately half that for ecdysone.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the presence of makisterone A in Drosophila larvae and the secretion of 20-deoxymakisterone A by the ring gland

Redfern

1984

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Ring glands or brain-ring gland complexes from third-instar Drosophila melanogaster larvae secreted ecdysone [(22R)-2j3,3,,14a,22,25-pentahydroxy-5i3-cholest-7-en-6-oneJ and two less polar ecdysteroids (LP1 and LP2) in vitro. Radioimmunoassay with different antisera indicated that LP1 and LP2 are side-chain-modified analogues of ecdysone. In high-performance liquid chromatography, the retention time of LP2 was equivalent to that of a precursor of makisterone A [(20R,22R)-2fi,3fi,l4a,20,22,25-hexahydroxy-24-methyl-5p-cholest-7-en-6-one] secreted by Dysdercusfasciatus prothoracic glands in vitro. LP2 was metabolized in vitro by the fat body of DrosophilM larvae to a product with the characteristics of iakisterone A when analyzed by gas chromatography/mass spectrometry (selected ion nionitoring). Evidence was obtained for the presence of mikisterone A in. Drosophila larvae. These data suggest that LP2 is 20-deoxymakisterone A (24-methylecdysone) and that makisterone A could function as an additional moulting hormone in Drosophila, although 20-deoxymakisterone A production is apparently dependent on the sterol composition of the diet. LP1 has not been identified.The insect steroid ecdysone, a C27 ecdysteroid [(22R)-2p,3,8 14a,22,25-pentahydroxy-5p-cholest-7-en-6-one], is secreted by the prothoracic glands (1) or, in cyclorrhaphous Diptera, by the ring gland (2). Ecdysone is hydroxylated at C-20 by monooxygenase enzyme systems in the fat body, gut, and Malpighian tubules to give the biologically active moulting hormone 20-hydroxyecdysone [(20R,22R)-2P,3,8,14a,20,22,25-hexahydroxy-5/3-cholest-7-en-6-oneI (3).Recently, the moulting hormone in Dysdercus fasciatus (4) and some other species of phytophagous Hemiptera (5) has been identified as a C28 ecdysteroid, makisterone A [(20R,22R)-2(,3(3,14a,20,22,25-hexahydroxy-24-methyl-5f3-cholest-7-en-6-one], which differs from 20-hydroxyecdysone in the presence of an additional methyl group at C-24 of the ecdysteroid side chain. By analogy with other insects (1), the prothoracic glands of these hemipterans would be expected to secrete the 24-methylated analogue of ecdysone, 20-deoxymakisterotle A (24-methylecdysone).In a previous study, it was shown that Drosophila ring glands secrete ecdysteroids in vitro and that the rate of secretion rea'ches a maximum towards the end of the last larval instar (6). Sarcophaga ring glands secrete ecdysone in vitro (2), and the experiments reported here were initially designed (7) were used.Yeast RNA was replaced with uridine and inosine, and lecithin was replaced with choline chloride (7). The medium constituents were purchased from Sigma [uridine, nicotinic acid, thiamine HCl, calcium pantothenate, pyridoxine, biotin, and ergosterol (ergosta-5,7,22E-trien-3,8-ol) Organ Culture. Ring glands or brain-ring gland complexes from third-instar Drosophila larvae with partially or fully bloated larval salivary glands were dissected and cultured (three to six ring glands or brain-ring gland complexes per culture) in Grace's medium (Flow La...

show abstract

Radioimmunoassay of arthropod moulting hormone: ß‐ecdysone antibodies production and ¹²⁵i‐iodinated tracer preparation

Cited by 58 publications

References 6 publications

Smt3 is required forDrosophila melanogastermetamorphosis

Smt3 is required forDrosophila melanogastermetamorphosis

Genes of the Ecdysone Biosynthesis Pathway Are Regulated by the dATAC Histone Acetyltransferase Complex in Drosophila

Evidence for the presence of makisterone A in Drosophila larvae and the secretion of 20-deoxymakisterone A by the ring gland

Contact Info

Product

Resources

About

Radioimmunoassay of arthropod moulting hormone: ß‐ecdysone antibodies production and 125i‐iodinated tracer preparation

Cited by 58 publications

References 6 publications

Smt3 is required forDrosophila melanogastermetamorphosis

Smt3 is required forDrosophila melanogastermetamorphosis

Genes of the Ecdysone Biosynthesis Pathway Are Regulated by the dATAC Histone Acetyltransferase Complex in Drosophila

Evidence for the presence of makisterone A in Drosophila larvae and the secretion of 20-deoxymakisterone A by the ring gland

Contact Info

Product

Resources

About

Radioimmunoassay of arthropod moulting hormone: ß‐ecdysone antibodies production and ¹²⁵i‐iodinated tracer preparation