Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin.

Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of exposure to (anti-)oestrogens and their mimics in fish [Sumpter, J.P., 1995. Feminized responses in fish to environmental estrogens. Toxicol. Lett. 82, 737-742; Arukwe, A., Goksøyr, A., 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp. Hepatol. 2, 4. ]. In some instances, links have been drawn between high level induction of Vtg and adverse health effects in fish [Herman, R.L., Kincaide, H.L., 1988. Pathological effects of orally administered estradiol to rainbow trout. Aquaculture 72, 165-172; Schwaiger, J., Spieser, O.H., Bauer, C., Ferling, H., Mallow, U., Kalbfus, W., Negele, R.D., 2000. Chronic toxicity of nonylphenol and ethinyloestraiol: haematological and histopathological effects in juvenile common carp (Cyprinus carpio). Aquat. Toxicol. 51, 69-78]. The widespread use of Vtg as a biomarker has led to the development of a variety of assays to quantitatively measure Vtg concentrations in tissue samples from fish, and hence a need for a standardization of the performance criteria and validation of such assays [Goksøyr, A., Eidem, J.K., Kristiansen, S.I., Nilsen, B.M., 2003. On the need for a standardized set-up for validation studies of fish vitellogenin assays as an endpoint in endocrine disruptor testing and screening-a proposal. ]. One of the most popular test fish species for assessing chemical effects is the fathead minnow (Pimephales promelas), which is now used widely for studies into endocrine disruption [Panter, G.H., Hutchinson, T.H., Lange, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., 2002. Utility of a juvenile fathead minnow screening assay for detecting (anti)estrogenic substances. Environ. Toxicol. Chem. 21, 319-326; Hutchinson, T.H., Yokota, H., Hagino, S., Ozato, K., 2003. Development of fish tests for endocrine disruptors. Pure Appl. Chem. 75, 2343-2353]. This paper describes the development and validation of a new, homologous enzyme-linked immunosorbent assay (ELISA) for quantification of Vtg in this fish species.

A novel, extraneuronal role for cyclin-dependent protein kinase 5 (CDK5): modulation of cAMP-induced apoptosis in rat leukemia cells.

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.

Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions.

The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.