Entrainment of circadian rhythm to a photoperiod reversal shows retinal dystrophy in RPE65(-/-) mice.

Light entrainment of circadian rhythms is mediated by classical "visual" photoreceptors (rods and cones) as well as "nonvisual" photoreceptive elements (light-detecting cells that do not contribute to classical "vision"). This paper aimed to assess whether light entrainment of locomotor circadian rhythms in mice with impaired rods and cones differs from normal controls and whether this technique, alongside existing techniques, could be used to assess visual function. The study was primarily interested in differences between the entrainment of circadian rhythms of normal-sighted C57Bl/6J mouse and the C57Bl/RPE65 knockout mouse (RPE65(-/-)), although C3H/HeJ (rd/rd) mice were included as a preexisting model of retinal degeneration. Circadian rhythms of motor activity before and after a 12-h light reversal were assessed in custom-built cages that continuously monitored movement. The controls showed a significantly higher mesor and amplitude when compared to the RPE65(-/-) and rd/rd mice. Despite the loss of rods and cones, the RPE65(-/-) and rd/rd maintained a 24-h circadian rhythm entrained to light similar to controls and were capable of circadian reentrainment to a 12-h light reversal. Importantly, this light reentrainment of the circadian phase occurred at a significantly slower rate in the retinal degenerate models than in the controls. The RPE65(-/-) model demonstrates a retinal degenerate reentrainment phenotype when compared to the rd/rd model. It is suggested that these retinal degenerate mice retain the ability to detect light for the purposes of circadian rhythm entrainment. However, alterations of specific parameters of the circadian rhythm with loss of rods and cones may provide measures of loss of visual function (sight).

Virtual kinetics: using statistical experimental design for rapid analysis of enzyme inhibitor mechanisms.

Modern automated drug-screening can generate hundreds of inhibitor leads from diverse chemical sources in a short period of time. Traditional methods of inhibitor analysis are resource intensive and limit the number of inhibitors that can be analyzed for their mechanism of inhibition. This paper presents methods we have developed for rapid estimation of both potency and mechanism of potential inhibitor leads for a biochemically complex screening target (protein kinase C) using commercially available computer programs for statistical experimental design. Our findings indicate that, with careful choice of factor levels, statistical experimental design clearly identifies the various interactions of the assay components with inhibitors. Suitably plotted, the data can be used to examine the competitive nature of the inhibitor and can provide estimates of IC50 and Michaelis constants useful for planning further kinetic work. The techniques used are amenable to automation and should be useful for identifying inhibitors that may have only marginal potency, but exhibit desirable mechanistic profiles suitable for structural analoging efforts.