Daily profile in melanopsin transcripts depends on seasonal lighting conditions in the rat retina.

The retinal photopigment melanopsin (Opn4) mediates photoentrainment of the circadian system. In the present study, seasonal regulation of the melanopsin gene was investigated in comparison with the arylalkylamine N-acetyltransferase (AA-NAT) gene as an indicator of retinal pacemaker output. For this purpose, the daily profiles in the amount of melanopsin mRNA and AA-NAT mRNA were monitored under 8 : 16 h light/dark, 12 : 12 h light/dark and 16 : 8 h light/dark photoperiods using real-time polymerase chain reaction analysis. We found that, under all of the lighting regimes, melanopsin and AA-NAT expression oscillated with a peak around dark onset and the middle of the dark phase, respectively. The lighting regime influenced both genes, but in an opposing manner. Under long photoperiods, the duration of peak expression was prolonged for melanopsin, whereas it was shortened for AA-NAT. Under constant darkness, the rhythm of mRNA was abolished for melanopsin, but persisted for AA-NAT whereas, under constant light, the rhythm of mRNA was abolished for both genes. Our findings suggest that, in contrast to the AA-NAT gene, the daily and photoperiod-dependent regulation of the melanopsin gene does not rely on a circadian oscillator but is directly illumination-dependent.

Evaluation of the concept of heterology in a monoclonal antibody-based ELISA utilizing direct hapten linkage to polystyrene microtiter plates.

A series of new heterologous haptens has been synthesized and used as coating haptens in an antigen-immobilized immunoassay with a monoclonal antibody against atrazine. Coating was achieved by covalently linking the different haptens to a glutaraldehyde network directly bound to the polystyrene surface of a standard 96-well microtiter plate. With the assay designed in the antigen-immobilized format with direct chemical linkage of the hapten to the solid polystyrene surface well-defined hapten densities were achieved in all experiments. The results of different experiments with different coating haptens were comparable. Using different heterologous haptens it appears that the concept of heterology is applicable in this case and can be used to enhance the sensitivity of an immunoassay in the coating-hapten format.

Fos-related antigen 2 (Fra-2) memorizes photoperiod in the rat pineal gland.

As the physiological role of fos-related antigen-2 (Fra-2) is largely unknown and since the pineal plays an important role in the photoperiodic control of the body, we have tested the hypothesis that Fra-2 expression is photoperiod-dependent and may be involved in imprinting photoperiod on the pineal gland and the body as a whole. To this end, we have investigated Fra-2 mRNA expression and Fra-2 protein expression under various light/dark (LD) cycles. A clear nocturnal increase occurs for both monitored parameters under all photoperiodic conditions studied. The level of Fra-2 protein expression clearly depends on photoperiod, because the amount of protein at dark onset and during the night negatively correlates with the length of the photoperiod. Further, high-phosphorylated Fra-2 isoforms are abundant under all photoperiods tested, with the exception of LD 20:4. Because Fra-2 phosphorylation depends on cGMP, a depressed cGMP response to adrenergic stimulation under LD 20:4 appears to explain this finding. We conclude that photoperiod is imprinted on Fra-2 in terms of both protein amount and protein phosphorylation in the rat pineal gland. This imprinting becomes fully manifest after about 7 days only, suggesting that a number of altered photoperiodic cycles are required for pineal Fra-2 to "learn" that the photoperiod has changed. Reportedly, Fra-2 limits expression of the enzyme iodothyronine deiodinase type II, which catalyzes the intracellular deiodination of thyroxine prohormone to the active 3,3',5-triiodothyronine. We have found that the extent of Fra-2 expression inversely correlates with the dII gene response to cAMP; hence the photoperiodic regulation of Fra-2 may affect the body by changing pineal thyroid hormone metabolism.

An immunoassay for terbutryn using direct hapten linkage to a glutaraldehyde network on the polystyrene surface of standard microtiter plates.

2-Aminobutylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (ABA-atrazine) has been synthesized and used as a coating hapten in an immunoassay with a monoclonal antibody against terbutryn. Coating was achieved by covalently linking ABA-atrazine to a glutaraldehyde polymer network directly bound to the polystyrene surface of a standard 96-well microtiter plate. The assay was carefully optimized. In particular, the coating hapten concentration had a strong effect on the ELISA sensitivity. By including a pre-incubation step a low test midpoint (IC50-value) of 0.130 microg L(-1) was achieved. As far as we are aware this is the most sensitive ELISA for terbutryn yet reported. The coating-hapten-format presented is proposed as generally applicable, because the glutaraldehyde-modified microtiter plate surface enables stable immobilization of a broad variety of coating haptens.

New developments in immunochemical water analysis down to 30 mul sample volume.

The determination of trace levels of pesticides like atrazine in water samples of small, restricted volumes is one of the future demands of environmental analysis. In a brief review existing chromatographic and immunochemical methods for atrazine are critically discussed. Then a simple rapid enzyme-linked immunosorbent assay (ELISA) using the tip of an inoculation needle as a solid surface is presented. The sample volume could be reduced to 30 mul. The assay had a centre of the test IC(50) of 0.12 mug l(-1) and permitted the characterisation of atrazine at levels of 0.022-2.90 mug l(-1). A first outlook for automatisation is given. The new method was compared with an ELISA using 96 well microtiter plates as a solid phase. Surface water samples with low atrazine contents were analysed to check the new method.