An unsuspected ecdysteroid/steroid phosphatase activity in the key T-cell regulator, Sts-1: surprising relationship to insect ecdysteroid phosphate phosphatase.

The insect enzyme ecdysteroid phosphate phosphatase (EPP) mobilizes active ecdysteroids from an inactive phosphorylated pool. Previously assigned to a novel class, it is shown here that it resides in the large histidine phosphatase superfamily related to cofactor-dependent phosphoglycerate mutase, a superfamily housing notably diverse catalytic activities. Molecular modeling reveals a plausible substrate-binding mode for EPP.

Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum.

The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of diverse aspects of cellular activity. Specifically, PK-A appears to play a key controlling role in the maturation of spermatids. Using a PCR-based approach, with degenerate primers from the highly conserved regions of the PK-A catalytic (C) subunit in combination with 5' and 3' RACE, we have cloned three cDNAs for the PK-A C-subunit of the male tick, Amblyomma hebraeum.

Expression and down-regulation of cytochrome P450 genes of the CYP4 family by ecdysteroid agonists in Spodoptera littoralis and Drosophila melanogaster.

The function of CYP4 genes in insects is poorly understood. Some CYP genes are up-regulated by ecdysteroids and a number of Cyp4 genes in Drosophila melanogaster have been shown by microarray to be down-regulated when the ecdysteroid titre is high, suggesting hormonal regulation. Here, we report the utilization of certain cloned CYP4 cDNAs/fragments to probe their developmental/tissue expression in the Lepidopteran, Spodoptera littoralis, including the effects of ecdysteroid receptor agonists (bis-acyl hydrazines).

Characterization in relation to development of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, in Lepidoptera.

Cytochrome P450 enzymes are involved in a number of steps in ecdysteroid (moulting hormone) homeostasis in insects. We report the cloning and characterization of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, from the cotton leafworm, Spodoptera littoralis. Northern blot analysis showed that the mRNA transcript was expressed at times of increasing ecdysteroid titre in final instar S.

Regulation of ecdysteroid signalling during Drosophila development: identification, characterization and modelling of ecdysone oxidase, an enzyme involved in control of ligand concentration.

The steroidal moulting hormones (ecdysteroids) mediate developmental transitions in insects, and their regulation is mainly controlled by the production and inactivation of these steroid hormones at the appropriate developmental times. One route of metabolism of ecdysteroids in insects involves EO (ecdysone oxidase)-catalysed conversion into 3-dehydroecdysteroid, which undergoes reduction to the corresponding 3-epiecdysteroid. By a twin-stranded bioinformatics approach, employing both phylogenomics and model structure-based analysis, we first predicted that DmEO (the EO of Drosophila melanogaster) corresponds to the protein product of gene CG9504.

A thrombin inhibitor from the ixodid tick, Amblyomma hebraeum.

A novel thrombin inhibitor named Amblin was identified from the haemolymph of the ixodid (hard) tick, Amblyomma hebraeum, and the coding cDNA was isolated from a tick cDNA library. This cDNA codes for a preprotein of 166 amino acids, including a predicted signal peptide composed of 15 amino acids N-terminal to the mature Amblin. The 151-amino-acid mature Amblin contains 14 cysteines and two Kunitz-like domains.

A new type of antimicrobial protein with multiple histidines from the hard tick, Amblyomma hebraeum.

A novel 11 kDa antimicrobial protein, named as hebraein, and having a unique amino acid sequence, was purified from the hemolymph of fed female Amblyomma hebraeum ticks. A full-length cDNA clone encoding hebraein was isolated from a cDNA library made from tick synganglia. Hebraein consists of 102 amino acids, including 6 cysteine residues; has 9 histidines in its C-terminal domain that are mainly present as HX repeats; and has no significant similarity to any known protein.

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans.

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing.

Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum.

Two non-cationic defensin-like antimicrobial peptides, named Amblyomma defensin peptide 1 and Amblyomma defensin peptide 2, were identified from the hard tick, Amblyomma hebraeum, by a combination of suppression subtractive hybridization for differentially expressed genes and proteomics. cDNA clones encoding each of these two defensin-like antimicrobial peptides were isolated from the differentially expressed cDNA library of the tick synganglia (central nervous system). The preproproteins deduced from the cDNA sequences each have 92 amino acid residues.

Molecular cloning and induction of nuclear receptors from insect cell lines.

Fragments of EcR and USP were cloned from two insect cell lines, Sf21 and High Five cells (derived respectively from Spodoptera frugiperda and Trichoplusia ni), using a PCR-based approach employing degenerate primers designed on the basis of conserved regions of nuclear receptors, together with 5'- and 3'-RACE. An additional orphan nuclear receptor, HR4 fragment, was cloned from High Five cells. Comparison of these fragments with Manduca sexta counterparts showed that the cloned SfEcR [ecdysone receptor (EcR) from Sf21 cells] had high similarity to MsEcR-B1, whereas the cloned SfUSP [ultraspiracle (USP) from Sf21 cells] and TnUSP (USP from High Five cells) matched more closely to MsUSP-2 than to MsUSP-1.