Margarethe Spindler-Barth

Chitin metabolism: a target for drugs against parasites.

Chitin is an important component of the exoskeleton of arthropods and of the egg shell in nematodes, but it does not occur in vertebrates. Therefore, it represents a useful target for drugs against ectoparasitic crustaceans, insects and endoparasitic nematodes. In this review we describe the basic characteristics of chitin, chitin synthesis and degradation and the hormonal regulation of chitin metabolism. Substances interfering with chitin metabolism like benzoylphenyl-urea derivatives but also some recently detected compounds are described. The necessity for a more detailed understanding of chitin metabolism and the establishment of better model systems, like e.g. chitin producing insect cell lines, is stressed and some examples are given in this review.

Insect cell lines as tools for studying ecdysteroid action

Summary Recent research concerning ecdysteroid-responsive and ecdysteroid-producing cell lines is reviewed. The advantages and limitations of cell lines of defined and undefined origin are considered with regard to their suitability for studies on molecular, physiological, morphological and developmental aspects of ecdysteroid action. The considerable potential for future studies involving insect cell lines is indicated.

Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster

Abstract Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate liganddependent dimerization. We conclude that the enhanced regulatory potential by liganddependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligandbinding pocket is modified by heterodimerization

Expression of EcR and USP in Escherichia coli: Purification and functional studies

The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP). EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects. The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) [3H]ponasterone A. Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PALI. Interestingly, binding to the PAL1 element was also observed for EcR homodimers. USP homodimers, in turn, preferentially bound to the direct repeat element DR1. When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B.

Analysis of transcriptional activity mediated by Drosophila melanogaster ecdysone receptor isoforms in a heterologous cell culture system

Ecdysteroid regulation of gene transcription in Drosophila melanogaster and other insects is mediated by a heterodimer comprised of Ultraspiracle (USP) and one of three ecdysone receptor (EcR) isoforms (A, B1 and B2). This study revealed that the EcR/USP heterodimer displays isoform‐specific capabilities. EcRB1 is normally induced with a form of USP that is missing its DNA‐binding domain (DBD), although potentiation by juvenile hormone (JH) III is reduced. The EcRA and B2 isoforms, however, display almost no response to ecdysteroids with the DBD− USP. A mutation, K497E, in the shared ligand‐binding domain of the EcR isoforms caused elevated EcRB2‐specific affinity for a canonical ecdysone response element. The effects of directed modification and mutagenesis offer a strategy for developing hypotheses and considerations for studying in vivo function.

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

Abstract The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acidbinding residue within the ligand-binding pocket, I323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.

Developmental Effects of a Chimeric ultraspiracle Gene Derived From Drosophila and Chironomus

Summary: The ultraspiracle (usp) gene encodes a nuclear receptor that forms a heterodimer with the ecdysone receptor (EcR) to mediate transcriptional responses to the insect steroid hormone, 20‐hydroxyecdysone (20HE). The responses ultimately elicit changes associated with molting and metamorphosis. Although Ultraspiracle (USP) is required at several developmental times, it is unclear whether USP plays stage‐specific roles in Drosophila. A chimeric transgene (d/cusp), produced by replacing the ligand‐binding domain (LBD) of Drosophila USP with the equivalent domain from another Diptera, Chironomus tentans, was tested for its ability to rescue Drosophila usp mutants from early larval lethality. A single copy of the d/cusp was sufficient to rescue transformants from several lines through larval development but they died suddenly during the late third instar. Additional doses of d/cusp were required to allow survival through the adult stage, but they did not restore a normal prepupal contraction. Thus, the arrest at the onset of metamorphosis apparently is caused by the impaired ability of the chimeric USP to mediate a stage‐specific function associated with the LBD. genesis 28:125–133, 2000.

The level of chitinolytic enzymes and ecdysteroids during larval-pupal development in Ephestia cautella and their modifications by a juvenile hormone analogue

The influence of the juvenoid methoprene on ecdysteroid titre and enzymatic activity of N-acetylglucosaminidase and chitinase in Ephestia cautella was examined. Treatment of the larvae in the early fourth larval instar prevents not only the normal rise in ecdysteroid titre but also the increase in activity of both enzymes. Treatment of pupae has no effect on either the ecdysteroid titre or on the enzymatic activities. Larvae treated with methoprene during the last quarter of the larval stage show intermediate values in ecdysteroid titre as well as in chitinolytic enzyme activities. A causal relationship between moulting hormone titre and activity of chitin-degrading enzyme is proposed.

Ecdysteroid resistant subclones of the epithelial cell line from Chironomus tentans (Insecta, Diptera). I. Selection and characterization of resistant clones.

SummaryChironomus tentans cells were cultured in the presence of gradually increasing concentrations of 20-OH-ecdysone or a nonsteroidal molting hormone agonist, the benzoylhydrazine RH 5992, for a period of about 2 yr. From these cultures, subclones were selected, which are resistant to up to 25 µM 20-OH-ecdysone according to morphological (changes in cell shape and cell arrangement) and physiological criteria (acetylcholinesterase induction, secretion of chitinolytic enzymes, thymidine incorporation). Some subclones, selected in the presence of 20-OH-ecdysone, are resistant only to molting hormone, but still respond to RH 5992 morphologically and biochemically, whereas subclones selected in the presence of the benzoylhydrazine showed no reaction neither to 20-OH-ecdysone nor to the hormone agonist. Hormone resistance is stable; 3 mo. after hormone withdrawal, resistant clones still do not respond to renewed exposure to 20-OH-ecdysone or RH 5992, respectively. Because in all resistant subclones tested so far all hormonally regulated responses known from sensitive cells were no longer detectable, it is assumed that the hormone signaling pathway itself is interrupted. Possible mechanisms of hormone resistance were discussed.

Chitin synthesis in insect cell lines

Abstract In contrast to a cell line from the codling moth ( Carpocarpsa palmonella ) and mouse pituitary cells, cell cultures of non-biting midges ( Chironomus tentans ) and Drosophila melanogaster incorporate glucosamine (GlcN) into chitin. GlcN and N -acetyl-glucosamine (GlcNAc) give higher incorporation rates compared to glucose and mannose. The polymerization product formed in these cells is stable in alkali and against proteolytic digestion, but is hydrolyzed by HCl and chitinase (EC 3.2.1.14) from Streptomyces griseus in a time-dependent manner, which results in formation of GlcNAc, (GlcNAc) 2 and (GlcNAc) 3 . The apparent Km and V max for GlcN incorporation were determined to be 82 μM and 232 pmol d −1 μg protein −1 , respectively. Incorporation of GlcN was suppressed by SIR 8514, nikkomycin, polyoxin D, which inhibit chitin synthesis in other systems.