Klaus-Dieter Spindler

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.

Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene

OBJECTIVE Androgen insensitivity syndromes (AIS) in subjects with 46,XY karyotype and normal or even elevated androgen blood levels are characterized by various aberrations in male differentiation and virilization. AIS is often accompanied by a broad spectrum of abnormal binding characteristics of the androgen receptor (AR). In order to investigate the correlation between the degree of virilization defect and the type of androgen binding abnormalities and/or the nature of the mutation in the AR gene, we determined androgen binding characteristics of the AR protein and the sequence of the AR gene in clinically and biochemically well characterized patients with various degrees of androgen resistance.

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.

Vesicle formation and ecdysteroid-induced cellular differentiation in the epithelial cell line from Chironomus tentans

Abstract The epithelial cell line from the midge Chironomus tentans grows in multicellular vesicles (Wyss, 1982) of 100 up to 1000 μm, depending on the culture conditions. These vesicles are formed by a cell monolayer. In contrast to the cyst formation in vertebrate cells (McAteer et al. , 1988) the vesicles originate from single cells by cell division and not by aggregation. The cells are arranged in a polar fashion: microvilli are found only at the outside of the vesicle (apex-out vesicles). The same types of cell junction, which are present in insect epithelia. are observed in Chironomus cells: zonulae adhaerentes, septate junctions, spot desmosomes and gap junctions. The intrusion of heavy dye molecules like Ruthenium red/OsO 4 into the lumen of the vesicles is prevented by septate junctions. After addition of 20-OH-ecdysone to preformed vesicles the simple low squamous epithelial cells change into columnar and cuboidal cells which show a stratified or a random arrangement. This is accompanied by an increase of the number of microtubules, which are sometimes arranged in bundles. They are frequently orientated along the main axis of the cells.

Electron microscopic demonstration of glucocorticoid recognition sites on isolated rat hepatocytes

Ultrastructural evidence is presented for the presence of membrane-bound glucocorticoid recognition and binding sites. Corticosterone was derivatized at 3 different positions and coupled covalently to bovine serum albumin (BSA). All three derivatives competed for binding of [3H]corticosterone by isolated rat hepatocytes. The most effective competitor, corticosterone-succinate-BSA (CSB), was adsorbed onto colloidal gold particles (CSB-gold, 17 +/- 3 nm dia). When isolated rat hepatocytes or mouse pituitary tumor cells (AtT 20) are incubated with CSB-gold, specific binding in the microvilli-rich region of these cells is seen. This binding of CSB-gold is reduced by about 50% in the presence of unlabelled CSB or corticosterone.

Characterization of cytoplasmic ecdysteroid receptors in the hypodermis of the crayfish, Orconectes limosus

[3H]Ecdysone, [3H]20-OH-ecdysone and [3H]ponasterone A were specifically bound by the 120 000 g supernatant of the homogenate from crayfish hypodermis. According to the HPLC test, the bound hormone was not metabolized. Analysis of the cytosol receptor by sucrose-density-gradient centrifugation, gel filtration and enzymatic degradation revealed the protein character of the receptor, a molecular weight of 70 000-80 000 D, and 5S. The binding capacity was rapidly lost after treatment with N-ethyl-maleimide or heating for 10 min at 60 degrees C. Optimal conditions for the demonstration of the receptor were incubation at 0-8 degrees C for 30 min and a salt concentration of 120 mM. A sequence of moulting hormones, and steroid hormones without moulting hormone activity were tested for ligand specificities. Estimation KD values yielded 3-6 x 10(-11) M for ponasterone A, 2-3 x 10(-9) M for 20-OH-ecdysone and 2 x 10(-8) M for ecdysone.

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na+/K+-ATPase

Isolated rat hepatocytes possess a saturable glucocorticoid uptake system with high affinity (Kd value = 2.8 +/- 0.7 x 10(-8) M; 318,000 +/- 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30-40% if the cells are incubated simultaneously with [3H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na+/K(+)-ATPase-inhibitors, ouabain and quercetine. These Na+/K(+)-ATPase-blockers exert half-maximal inhibition at 3 x 10(-7) and 3 x 10(-6) M, respectively. A slight increase in K+ concentration and a corresponding decrease in Na+ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5 alpha- and 5 beta-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.

Structural and developmental analysis of a gene cloned from the early ecdysterone-inducible puff site, I-18C, in Chironomus tentans.

A gene (I-18C) cloned from the early ecdysterone (EDS)-inducible puff site, I-18C, in Chironomus tentans salivary glands, codes by differential processing for at least five transcripts which can be grouped into spliced and unspliced transcripts. The spliced group comprises several RNAs of approx. 4.6 kb, and the unspliced group comprises the 1.8-kb and 6.5-kb RNAs. All these transcripts have a similar or the same transcription start point. The steady-state level of the spliced 4.6-kb RNAs reveals some parallels with EDS concentrations in animals during the various developmental stages analyzed, while the levels of the unspliced 1.8-kb and 6.5-kb RNAs do not correlate with the EDS concentrations.

DNA-binding properties of an “ecdysteroid receptor” from epithelial tissue culture cells of Chironomus tentans

Recently an ecdysteroid binding protein (“ecdysteroid receptor”) has been identified in extracts of epithelial tissue culture cells of the dipteran insect Chironomus tentans. We now show that this protein also displays high affinity to DNA. When using a solid phase binding assay an association rate constant of 3.3 × 106 M−1 s−1 and a dissociation rate constant of 2 × 10−4 s−1 were determined for the ecdysteroid receptor-immobilized DNA complex at 0°C. An apparent equilibrium dissociation constant of 6.1 × 10−11M could be estimated from these values indicating a high affinity binding of ecdysteroid receptors to immobilized DNA. The binding of ecdysteroid receptors to immobilized DNA was saturable. Only double stranded free DNA competed significantly for ecdysteroid receptor binding to immobilized DNA. Furthermore, DNA sequences that contain ecdysone response elements (EcREs) from hormonally regulated Drosophila genes or sequences from an ecdysteroid-inducible gene of C. tentans revealed a 10–350 times higher affinity to ecdysteroid receptors than random sequences of calf thymus DNA or from a plasmid.