Ecdysteroid Activity Research Articles

Monitoring ecdysteroid activities using genetically encoded reporter gene in Daphnia magna

Ecdysteroid is an important hormone that regulates growth, reproduction, and embryogenesis in arthropods. However, little is known about its role and action mechanism in crustaceans, despite their pivotal role in aquatic ecosystem. Daphnia magna, a freshwater crustacean, is used as a classic model organism in ecology and ecotoxicology. Its ecdysteroid activity has been partially characterized previously. However, the spatio-temporal behavior of ecdysteroid, especially during early embryogenesis, when it is thought to have pivotal roles, is still unclear. Thus, we proposed a genetic modification approach by integrating a reporter gene exhibiting ecdysteroid activity in vivo.We used the clustered regularly interspaced palindromic repeats (CRISPR) genome editing technique, followed by non-homologous end-joining (NHEJ) pathway as the transgenesis method to generate the ecdysteroid reporter transgenic Daphnia. One transgenic Daphnia containing one copy of the ecdysone response element (EcRE)-controlled reporter gene mCherry was successfully obtained and was designated EcRE-mCh.The expression of mCherry was observed during early embryogenesis starting from 12 h after ovulation (hao). The time-lapse imaging during 12–24 hao showed the growing expression of mCherry signal originating from the posterior section of embryo and then migrating toward the anterior section. From 18 hao, the signal was detected around the developing thoracic appendages and localized between the first to third thoracic segments. The establishment of this EcRE-mCh line and its ability to exhibit ecdysteroid activity spatio-temporally might serve as convenient tool to elucidate the roles of ecdysteroid during the early stage of animal development. Moreover, the expression of mCherry in response to the presence of ecdysteroid in water suggests that EcRE-mCh could be used for monitoring ecdysteroid activities in environmental water.

Backstabbing P-gp: Side-Chain Cleaved Ecdysteroid 2,3-Dioxolanes Hyper-Sensitize MDR Cancer Cells to Doxorubicin without Efflux Inhibition.

P-glycoprotein (P-gp, ABCB1) over-expression, causing a multi-drug resistant (MDR) phenotype, is a major problem in cancer chemotherapy that urgently requires novel approaches. Our previous studies showed certain ecdysteroid derivatives as promising chemo-sensitizers against MDR and non-MDR cancer cell lines while also exerting mild to moderate inhibition of P-gp function. Here we report the preparation of a set of substituted 2,3-dioxolane derivatives of poststerone, a known in vivo metabolite of 20-hydroxyecdysone (20E). In contrast with previously studied ecdysteroid dioxolanes, the majority of the new compounds did not inhibit the efflux function of P-gp. Nevertheless, a strong, dose dependent sensitization to doxorubicin was observed on a P-gp transfected cancer cell line and on its susceptible counterpart. We also observed that the MDR cell line was more sensitive to the compounds’ effect than the non-MDR. Our results showed for the first time that the chemo-sensitizing activity of ecdysteroids can be fully independent of functional efflux pump inhibition, and suggest these compounds as favorable leads against MDR cancer.

The molecular and physiological impact of bisphenol A in Sesamia nonagrioides (Lepidoptera: Noctuidae).

In the present study we investigated the potential relative effects of bisphenol A (BPA) and RH-5992 (tebufenozide) on the development and metamorphosis of the corn stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). A number of morphological and molecular factors were examined in order to identify the toxic and the endocrine-relative action of these two chemicals. We observed that BPA, RH-5992 and the combination of BPA/RH-5992 caused a developmental delay by extending the transition period between larval and pupal instars. These chemicals also reduced adult emergence and caused molting malformations during development and metamorphosis. In the corn stalk borer, BPA exhibits ecdysteroid activities in a fashion similar to that of the ecdysone agonist RH-5992. These results suggest that exposure to environmentally relevant concentrations of BPA during the early stages of the corn borer's life cycle can result in various disorders that may be a consequence of endocrine disruption. The molecular mechanism by which BPA interferes with the physiological processes was also investigated. A significant induction was observed in the expression levels of the ecdysone-induced genes SnEcR and SnUSP, after injection of BPA and RH-5992. Additionally, we found that BPA acts as a very weak agonist of ecdysteroids in Bombyx mori derived Bm5 cell lines. From these cellular and molecular assays, our results brought evidence that BPA, like RH-5992, interferes with the ecdysteroidal pathways of the lepidopteran insect species.

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal mode analysis

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50=3.3μM) and Bm5 cells (EC50=5.3μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71μM and 0.00089μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039μM; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18μM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.

Ecdysteroid 화합물들의 화학구조 특성에 대한 비교연구

Objectives: This study was conducted in order to investigate the specific correlation between physicochemical properties and bioactivity in ecdysteroids found in living organisms. Methods: The examined steroidal compounds were classified into three groups according to their relevance to ecdysone activity. Each compound molecule was completely drawn to automatically calculate its physicochemical parameters and docked against 20-hydroxyecdysone to calculate the total distance. Electronic charge distribution was also observed for each molecule. All procedures were conducted using a computational chemistry program. Results: Ecdysone agonists showed different ranges of parameter values, such as log P, hydrophilic-lipophilic balance (HLB), solubility parameter (SP), hydrophilic surface (HPS), hydrogen bond (HB) and Kappa 2, when compared with antagonists and steroids without ecdysone activity. They also showed a similar electronic charge distribution that is significantly different from the electron charge distribution of antagonists and steroids without ecdysone activity. The total distance values of agonists, estimated by docking them with 20-hydroxyecdysone, were relatively small but showed no correlation with binding affinity with receptor ligand. Conclusions: These results suggest that physicochemical properties such as steric and electronic effects, hydrophobicity and hydrogen bonding may operate in combination to determine the binding activity of ecdysteroids to the receptor protein.

Effect of total ecdysteroid preparation from Silene viridiflora on the immune state of experimental animals under normal and secondary immunodeficiency conditions

The isolation of total ecdysteroids from Silene viridiflora is described. The preparation acts as an effective immunomodulator in normal animals (mice, 18 – 20 g) and in mice with secondary immunodeficiency developed under irradiation, acute toxic hepatitis, and prolonged physical load conditions. The immunomodulating activity of total ecdysteroids from S. viridiflora is comparable with that of the known immunity stimulator T-activin, a preparation of polypeptide nature from cattle thymus.

Effects of atrazine and endosulfan sulphate on the ecdysteroid system of Daphnia magna

The ecdysteroid system is used by crustaceans and other arthropods as the major endocrine signalling molecules, regulating processes such as molting and embryonic development. The aim of this study was to evaluate the ecdysteroid activity of two pesticides (atrazine and endosulfan sulphate), with distinct modes of action and which act in the juvenoid system of the crustacean Daphnia magna as weak juvenoid compounds . To assess the ecdysteroid activity, we first exposed maternal daphnids and embryos to nominal concentrations of the pesticides and determined the effect promoted by these pesticides on the molting frequency and on abnormalities in the embryos development. Furthermore, we evaluated if the toxic effects observed with the isolated pesticides were promoted or not by the disruption of the ecdysteroids system of the crustacean, by co-administrating of them with 20-hydroxyecdysone hormone. This hormone is the prime form of the invertebrate’s ecdysteroids system and it is responsible for the crustacean molting process. Both pesticides induced an increase of embryo abnormalities development. Endosulfan sulphate promoted a delay in the molting process. The effects induced by atrazine were not altered by co-exposure to 20-hydroxyecdysone. In contrast, the co-administration of 20-hydroxyecdysone allowed the reversion of the effects on both the molting process and embryonic development elicited by endosulfan sulphate. These results suggest that atrazine promotes its toxicity without interfering with the ecdysteroid activity of the crustacean. On the contrary, endosulfan sulphate is an anti-ecdysteroidal compound for D. magna.

In vitro cultures of pupal integumental explants to bioassay insect growth regulators with ecdysteroid activity for ecdysteroid amounts and cuticle secretion

The present study evaluated the in vitro bioassay with pupal integumental explants of the mealworm Tenebrio molitor to test the activity of two types of insect growth regulators (IGRs) with ecdysteroidal action. We assessed the imidazole derivative KK-42, that is know as ecdysteroid biosynthesis inhibitor, and the dibenzoylhydrazine RH-0345 (halofenozide) that representing a novel group of IGRs with ecdysteroid agonist action. Two biological endpoint were used, namely the production of ecdysteroid hormone and cuticle secretion in vitro. Also the test was done with and without renewment of the culture medium to test the persistence of action of each compound. Essentially, RH-0345 provoked higher ecdysteroid amounts, while KK-42 a reduction. Interestingly when KK-42 was followed by RH0345, the inhibitory effect was compensated. In parallel, the deposition of cuticle in vitro was assessed and it was clear that RH-0345 could induce apolysis premature in the integument explants with the formation of a new cuticle. Here the protein and chitin contents of the explants were tested with RH0345 and three analogous compounds, RH-2485, RH-5992 and RH-5849.

Isolation of unique ecdysteroids from the Ajuga reptans var. reptans

Ecdysteroids represent biologically active steroids which attract great scientific interest because of their perspective use in the conventional health improvement methods and they have a tremendous potential in the most modern therapies (gene-switch systems), too. Several studies proved the activity of ecdysteroids to increase the rate of protein synthesis in mammalian tissue without any hormonal consequences. Because of their anabolic activity, ecdysteroids and ecdysteroid- containing preparations are widely advertised on the Internet as growth promoters. The use of ecdysteroids as inducers is also promising in genetics.

Antimicrobial activity of natural ecdysteroids from Serratula coronata L. and their acyl derivatives

Natural ecdysteroids isolated from Serratula coronata L., including 20-hydroxyecdysone (20-HE), 25-S-inokosterone, and ecdysone, did not exhibit antimicrobial activity with respect to most standard test microbe cultures. The introduction of the acetyl group into the 20-HE molecule significantly increased the antibacterial activity with respect to microbes inducing inflammatory and purulent processes. Acyl derivatives of 20-hydroxyecdysone can be suggested for use in wound-healing compositions

Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2)

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose–response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.

Action of 24-epibrassinolide on a cell line of the beet armyworm,Spodoptera exigua

The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.

Low-Polarity Phytoecdysteroids from the Juice of Serratula coronata L. (Asteraceae)

Eleven phytoecdysteroids have been isolated and identified from the low polarity fractions of Serratula coronata juice by a combination of column chromatography and HLPC. Four of them are new, i.e. ecdysone 22-acetate 7, (25S)-inokosterone 26-acetate 9, 20,22-O-(R-ethylidene)-20-hydroxyecdysone 10 and 20,22-O-(R-ethylidene)-ajugasterone C 11. The biological activity of these new ecdysteroids in the Drosophila melanogaster BII cell bioassay is reported.

Development of a cell-based screening assay for invertebrate molting disruption

The use of transgenic cell lines for relatively rapid, sensitive and reproducible assays for the detection and semi-quantitative measurement of contaminants in environmental media has increased in recent years. For example, many accepted assays rely on the arylhydrocarbon receptor interaction to screen for dioxins and related compounds in environmental samples. However, these systems are poorly developed or absent in lower organisms, and since arylhydrocarbon receptor interaction is a necessary early step in the development of dioxin toxicity, dioxins are relatively non-toxic to invertebrates. As a result, assays based on this interaction have no relevance for assessing the risk of environmental contamination to these organisms at the base of all food chains. Alternatively, arthropods possess highly developed ecdysone receptor systems that can be used similarly to the arylhydrocarbon receptor system as the basis of an assay with high relevance for these important classes of invertebrates. A new transgenic cell line was developed in order to create a rapid assay for ecdysone interactions to detect and measure the activity of environmental contaminants that are chronically toxic to lower organisms, specifically the invertebrate phylum Arthropoda. The cell line is based on Invitrogen's® Ecdysone-Inducible Mammalian Expression System, which consists of two plasmids, one of which expresses the heterodimeric ecdysteroid receptor while the other contains the receptor-ligand response element E/GRE. When ecdysone or another ligand having ecdysteroid activity is present, the binding of the receptor-ligand complex to the response element results in transcription of the reporter gene (β-galactosidase), the activity of which can be monitored colorimetrically. The plasmids were stably transfected into HepG2 human liver cells. Preliminary results show that Aroclor 1242, which is known to inhibit molting in invertebrates, also inhibited molting in juvenile crawfish (Procambarus clarkii) at 100 μg l−1 and normal ecdysteroid response in this new transgenic cell line, HepG2-EcR, at similar concentrations indicating that this new assay shows promise for future use as a screening tool.

Developmental toxicity of testosterone in the crustacean Daphnia magna involves anti-ecdysteroidal activity

Testosterone has been shown to cause developmental arrest of embryonic daphnids ( Daphnia magna). The present study was undertaken to determine whether this toxicity might be due to anti-ecdysteroidal activity associated with testosterone. The effect of testosterone on molt frequency of early instar daphnids was first evaluated to determine whether testosterone interfered with this ecdysteroid-regulated process. Molt frequency was delayed by exposure to testosterone and this effect was mitigated by co-exposure to the ecdysteroid 20-hydroxyecdysone. Testosterone exposure concentrations that interfered with molting also elicited developmental abnormalities among neonatal organisms produced by maternal organisms that were continuously exposed to testosterone or among embryos that were removed from unexposed mothers and exposed directly to the hormone. Embryos were significantly protected against the developmental toxicity of testosterone by co-exposure to 20-hydroxyecdysone. Taken together, these results demonstrated that the embryo toxicity of testosterone to daphnids is due largely to its ability to interfere with ecdysteroid control of development. Experiments next were conducted to determine whether testosterone interfered with ecdysteroidal activity by acting as an ecdysone receptor antagonist or by reducing endogenous ecdysone levels. Testosterone significantly antagonized the action of 20-hydroxyecdysone in an ecdysone-responsive cell line. Testosterone had no discernable effect on endogenous ecdysone levels in daphnids. These results demonstrated that (1) ecdysteroids regulate critical processes in daphnid embryo development, (2) testosterone elicits embryo toxicity to daphnids by interfering with ecdysteroid activity, and (3) ecdysteroid receptor antagonism could be one mechanism by which testosterone elicits these effects.

Species Specificity of Changes in Ecdysteroid Metabolism in Response to Ecdysteroid Agonists

Administration of the nonsteroidal ecdysteroid agonist RH-5849 or 20-hydroxyecdysone to final larval instar tobacco hornworm, Manduca sexta, has been shown to induce an ecdysteroid inactivation enzyme, ecdysteroid 26-hydroxylase, and the cytosolic inactivation enzymes ecdysone oxidase and ecdysteroid phosphotransferase. In this work, we show that induction of ecdysteroid 26-hydroxylase by the ecdysteroid agonists RH-5849, RH-5992 (tebufenozide), and RH-0345 (halofenozide) appears universal in lepidopteran species that show susceptibility to the agonists. Interestingly, the waxmoth, Galleria mellonella, which shows very low susceptibility to the agonists but whose ecdysteroid receptor is capable of binding the agonist, shows no induction of a 26-hydroxylase activity. It appears that the more potent ecdysteroid agonists in Lepidoptera, RH-5992 and RH-0345, show in general a greater induction of 26-hydroxylase than RH-5849. Feeding RH-5849 to the dipteran housefly Musca domestica results in the induction of an ecdysteroid phosphotransferase. The low toxicity of these ecdysteroid agonists in orthopteran and coleopteran orders also correlates with a lack of induction of ecdysteroid 26-hydroxylase activity. We propose that in species where ecdysteroid agonists are effective in stimulating an untimely premature molt, a response to a state of hyperecdysonism elicited by the agonists is induction of enzymes of ecdysteroid inactivation.

Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

In vivo and in vitro effects of tebufenozide and 20-hydroxyecdysone on chitin synthesis

Last-instar larvae of the beet armyworm, Spodoptera exigua, fed on diet containing the nonsteroidal molting hormone agonist, tebufenozide, at 1 mg/liter, exhibited a premature induction of chitin synthesis. Treatment with tebufenozide apparently caused normal amounts of chitin to be formed in the cuticle. This effect complemented the precocious induction of larval molting (apolysis) that was observed in response to tebufenozide. Subsequently, normal ecdysis was inhibited and treated larvae died while still within their old cuticle. In another series of experiments, 1-day-old pupae of the European corn borer, Ostrinia nubilalis, were injected with various doses of tebufenozide or 20E. Incorporation of N-[acetyl-1-14C]glucosamine served as a measure of chitin production and was monitored in cultured day-4 and day-5 claspers. Tebufenozide at 100, 200, and 400 ng induced synthesis in day-4 claspers and incorporation was enhanced 8-fold as compared to control claspers. A similar stimulation of chitin synthesis was observed with 2,000 ng of 20E. For day-5 claspers, high doses of tebufenozide inhibited chitin synthesis. Treatment with tebufenozide had a deleterious effect on adult development and even at the lowest dose injected, 10 ng, adult eclosion was completely inhibited. Our current results indicate that in both larvae and pupae/pharate adults, tebufenozide can induce the premature synthesis of chitin as can 20E, but the effective dose of tebufenozide is much lower than that for 20E. The results support the view that tebufenozide, although nonsteroidal in nature, is able to mimic the activity of ecdysteroids in inducing chitin synthesis. However, development in treated insects is abnormal and the newly formed cuticle is imperfect and unable to survive a molt. Arch. Insect Biochem. Physiol. 41:33–41, 1999. © 1999 Wiley-Liss, Inc.

Biological activity of natural and synthetic ecdysteroids in the BII bioassay.

The potencies of 19 ecdysteroids are compared in the BII bioassay, which reflects the affinity of binding to the ligand binding site of the Drosophila melanogaster ecdysteroid receptor. The compounds tested represent either natural products isolated from plants (phytoecdysteroids) or fungi (mycoecdysteroids) or synthetic analogues based on insect metabolites (zooecdysteroids). None of the tested compounds showed any antagonistic activity, but all possessed quantifiable agonistic activity. All the mycoecdysteroids were less potent than the major insect ecdysteroid, 20-hydroxyecdysone (20E). Also, conjugation of 20E with a glucose moiety results, as expected, in considerable reduction in biological activity, but the remaining activity is dependent on the position of conjugation. The implications of these findings for the structure/activity relationship of ecdysteroids are discussed.

Biological activity of natural and synthetic ecdysteroids in the B11 bioassay

The potencies of 19 ecdysteroids are compared in the B11 bioassay, which reflects the affinity of binding site of the Drosophila melanogaster ecdysteroid receptor. The compounds tested represent either natural products isolated from plants (phytoecdysteroids) or fungi (mycoecdysteroids) or synthetic analogues based on insect metabolites (zooecdysteroids). None of the tested compounds showed any antagonistic activity, but all possessed quantifiable agonistic activity. All the mycoecdysteroids were less potent than the major insect ecdysteroid, 20-hydroxyecdysone (20E). Also, conjuration of 20E with a glucose moiety results, as expected, in considerable reduction in biological activity, but the remaining activity is dependent on the position of conjugation. The implications of these findings for the structure/activity relationship of ecdysteroids are discussed. Arch. Insect Biochem. Physiol. 35:219–225, 1997. © 1997 Wiley-Liss, Inc.