ABSTRACT
Mst57Dc has been isolated as a male accessory gland transcript of Drosophila melanogaster. Its product is a secretory protein, which is phosphorylated by protein kinase A. In the present study, the expression pattern of Mst57Dc was analyzed. It is preferentially expressed in but not restricted to the male accessory glands. Other than in the accessory glands, it is slightly expressed in other body parts, including the head and female body. In the accessory glands, a high level of expression was detected right after eclosion when the titer of juvenile hormone III (JHIII) reaches a peak. Its accumulation was increased by mating, which has been known to act via JH. In ap56f a JH-deficient mutant, the level of Mst57Dc transcripts was about 60% of the wild type. Moreover a JH-responsive element like palindromic sequence and several sequence motifs were found in the 5' and 3' flanking regions of Mst57Dc. Taken together, JH is proposed as a regulator of Mst57Dc gene expression.
Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Gene Expression Regulation , Genes, Insect , Insect Proteins/genetics , Peptides/genetics , Phosphoproteins/genetics , Sesquiterpenes/pharmacology , Aging/genetics , Animals , Base Sequence , Drosophila melanogaster/embryology , Drosophila melanogaster/physiology , Female , Intercellular Signaling Peptides and Proteins , Male , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Sesquiterpenes/metabolism , Sexual Behavior, AnimalABSTRACT
The prothoracicotropic hormone (PTTH) of Drosophila melanogaster is a modulator of ecdysteroid (molting hormone) synthesis and was isolated and characterized from extracts of whole larvae (approximately 4 x 10(5) larvae). The purification protocol included delipidation, salt-extraction, heat treatment, conventional column chromatography, and HPLC, and yielded about 50 microg of pure hormone. Biological activity was followed using a ring gland in vitro assay in which ecdysteroidogenesis by control ring glands as measured by radioimmunoassay was compared with ring gland incubations containing active fractions. The molecular weight of the purified PTTH was 45 kDa and N-terminal amino acid sequence analysis indicated that those analyzed sequences displayed no significant homology with known peptides or peptide hormones, including PTTH from the silkmoth, Bombyx mori. Western blot analysis indicated that the native form of Drosophila PTTH was a single 66-kDa polypeptide with N-linked carbohydrate chains and intrachain disulfide bonds. The purified 45-kDa peptide is the deglycosylated form, a result of glycosidase activity present during preparation of the PTTH extract. The deglycosylated form shows heterogeneity, presumably as a result of varying degrees of deglycosylation at the N terminus.
Subject(s)
Drosophila melanogaster/chemistry , Glycoproteins/isolation & purification , Insect Hormones/isolation & purification , Nerve Tissue Proteins/isolation & purification , Animals , Biological Assay , Bombyx , Cross Reactions , Endopeptidases/pharmacology , Glycoproteins/pharmacology , Glycoside Hydrolases/pharmacology , Immunohistochemistry , Insect Hormones/pharmacology , Larva/chemistry , Nerve Tissue Proteins/pharmacology , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Sequence Analysis , Species SpecificityABSTRACT
Double quantum selective coherence transfer proton NMR spectroscopy has been used to observe glutathione in whole blood. The efficient water suppression of this technique avoids the need to resuspend the cells in D2O, hence avoiding equilibrium and kinetic isotope effects. Using this method we estimate the concentration of glutathione in fresh whole rabbit blood at approximately 1.7 mM.
