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Effect of insect hormones on RNA polymerases of mass-isolated imaginal discs of Drosophila melanogaster cultured in vitro.

Four chromatographically separable DNA-dependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) were partially purified from imaginal discs of Drosophila melanogaster. Their properties are similar to those described for RNA polymerases I and II isolated from other eukaryotes. In vitro incubation of discs with beta-ecdysone, juvenile hormone, or cycloheximide resulted in increased activity of RNA polymerase I. The increase was irreversible with beta-ecdysone incubation and removal but reversible with juvenile hormone incubation and removal. With beta-ecdysone, the rate of the increase in polymerase I activity paralleled the kinetics of ecdysone binding to discs and increases in the rate of precursor incorporation into RNA. A model to explain the increased acticity of RNA polymerase I is presented.

Amanitins

13C-NMR assignments of some insect molting hormones.

13C-NMR assignments of some insect molting hormones including ecdysone, 20-hydroxyecdysterone, ponasterone A and inokosterone, were made by using ecdysone as the model compound, whose carbon chemical shifts were assigned via NMR techniques including nOe and COSY, hetero-COSY and hetero-long range-COSY.

Ecdysone

A monoclonal antibody to the insect prothoracicotropic hormone.

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.

Animals

Insect peptide hormones, an overview of the present literature.

A comprehensive overview of the recent state of the art of insect peptide hormones with chemical structures is presented. An increased interest in insect neuropeptides and dynamic development of that research area has been influenced by a rapid improvement of instrumentation necessary for isolation and structural characterization. Several research teams have studied the relationships between biological properties of insect and vertebrate peptide hormones. Thus hormones from the AKH family can be considered glucagon counterparts, whereas the myotropic hormones such as proctolin and Lem-PK (LPK) are a substance P equivalent. Insect melanization hormones Bom-MRCH in their structural characteristics and properties resemble those of mammal MSH, and leucosulfakinins Lem-SK-I and -II show some similarities with gastrin II and cholecystokinin. Bombyxin-II (Bom-PTTH-II) reveals a structural homology with human insulin and similar biological properties to adenocorticotropic mammal hormone. Allatostatin (Dip-JHS-I) may be compared to somatostatin as it can be inferred from the observations that this peptide modulates JH secretion in cockroach, Blattella germanica. Determination of the primary structure of eclosion hormones Mas-EH and Bom-EH-II as well as the amino acid sequence of allatotropin and allatostatin is a significant contribution to the understanding of the molecular mechanisms of metamorphosis and insect development.

Amino Acid Sequence

Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate.

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and 1-oleyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate and diolein are inactive. The inhibitory effect of phorbol 12-myristate 13-acetate is fully reversed by 2E,6E-farnesol or by 2E,6E-farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20-hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.

Animals

A partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity.

A partition assay was developed to measure insect juvenile hormone (JH) I and III metabolism in biological samples containing both JH esterase and JH epoxide hydrolase activity. The assay utilizes commercially available radiochain 3H-labeled JH as substrate and the selective JH esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone. JH partitions into an isooctane phase and the metabolites JH acid, JH diol, and JH diol-acid into aqueous methanol after incubation of JH substrate with inhibited and uninhibited sample. The assay provides a time- and cost-efficient alternative to the currently available thin-layer chromatography method for the measurement of JH esterase and epoxide hydrolase activity.

Acetone

The activity of insect juvenile hormone mimics in larval Amblyomma hebraeum Koch (Acarina: Metastriata: Ixodidae).

A total of 14 insect juvenile hormone mimics was tested for activity in Amblyomma hebraeum by exposing newly engorged larvae to filter paper impregnated with hormone mimics. The most active compounds used in this assay were HS 103 (6-ethyl-3-pyridyl geranyl ether; EC50=0,0018 mg/cm2), ZR 512 (Ethyl 3,7,11-trimethyldodeca-2,4-dienoate; EC50=0,0022 mg/cm2), HS 2 (6-methyl-3-pyridyl geranyl ether; EC50=0,0035 mg/cm2), ZR 615 (N-ethyl 3,7,11-trimethyldodeca-2,4-dieneamide; EC50=0,0035 mg/cm2), ZR 777 (Prop-2-ynyl 3,7,11-trimethyldodeca-2,4-dienoate; EC50=0,0039 mg/cm2) and ZR 515 (Isopropyl-11-methoxy 3,7,11-trimethyldodeca-2,4-dienoate; EC50=0,0094 mg/cm2). Activity in this assay was similar to that reported in insects and was consistent with the susceptibility of these compounds to metabolic inactivation. The results suggest that ZR 615 may be of possible use in tick control.

Animals