Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening.

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.

Characterization of fatty acid synthase activity using scintillation proximity.

FAS is a 544-kDa dimeric enzyme consisting of seven functional catalytic components that synthesize long-chain fatty acids from acetyl-CoA and malonyl-CoA using NADPH as a cofactor. We have developed a novel radiometric, homogeneous procedure that directly detects FAS activity. The assay determines incorporation of [(3)H]acetyl-CoA into palmitic acid as catalyzed by FAS from rat liver. Radiolabeled palmitic acid is captured on a 384-well phospholipid-coated microtiter plate and is brought into close proximity with embedded scintillant, stimulating the emission of photons. Because it uses acetyl-CoA and malonyl-CoA as substrates, the procedure mimics the classical reductase assay. However, this method eliminates such labor-intensive steps as organic extraction, aspirations and washes, phase separations, and sample transfers. Furthermore, it offers advantages over photometric and fluorometric methods that indirectly measure FAS activity via NADPH absorbance. We present here kinetic and inhibition data for FAS using scintillation proximity. The assay is shown to be robust and reproducible.

Behavioral investigation of some possible effects of the central olivocochlear pathways in transgenic mice.

This study investigated the auditory behaviors of transgenic mice with deletions of alpha9 nicotinic acetylcholine receptor subunits. In the normal mammalian cochlea, the mechanical properties of outer hair cells are modified by the release of acetylcholine from olivocochlear efferent terminals. Electrophysiological correlates of this efferent feedback have not been demonstrated in alpha9 knockout mice, presumably because they are mediated by alpha9 receptors. Previous studies have associated lesions of olivocochlear pathways with hearing impairments in background noise. The prediction that alpha9 knockout mice would show similar deficits was tested by collecting psychophysical thresholds for tone detection and intensity discrimination from knockout mice, within-strain control subjects, and CBA/CaJ mice. Comparable performance was observed for the subject groups in quiet and in continuous background noise. The preservation of auditory function in alpha9 knockout mice suggests that central efferent pathways work in combination with the peripheral olivocochlear system to enhance hearing in noise, and may compensate for profound manipulations of peripheral feedback in highly routine testing procedures. An intriguing possibility is that these central mechanisms include the brainstem collaterals of olivocochlear neurons since their post-synaptic targets do not express alpha9 receptors and therefore are likely to maintain their effects in alpha9 knockout mice.

Contextual Treatment of Dissociative Identity Disorder.

Evidence for the effectiveness of contextual therapy, a new approach for treating adult survivors of prolonged child abuse (PCA), is provided via case studies of three women with Dissociative Identity Disorder (DID). Contextual therapy is based on the premise that it is not only traumatic experiences that account for PCA survivors' psychological difficulties. Even more fundamentally, many survivors grow up in an interpersonal context in which adequate resources for secure attachment and acquisition of adaptive living skills are not available. As a result, they are left with lasting deficits that undermine not only their current functioning, but also their ability to cope with reliving their traumatic memories in therapy. The primary focus of this treatment approach, therefore, is on developing capacities for feeling and functioning better in the present, rather than on extensive exploration and processing of the client's trauma history or, in the case of DID, of identity fragments. Treatment of the three cases presented ranged from eight months to two and one-half years' duration, and culminated in very positive outcomes. The women's reports of achievements, such as obtaining and maintaining gainful employment, greater self-sufficiency, and the establishment of more intimate and gratifying relationships, indicated marked improvements in daily functioning. Objective test data obtained at admission and discharge, and in one case, at follow-up, documented substantial reductions in dissociative, posttraumatic stress, depressive, and other symptoms.

A pilot study of cognitive behavioral stress management effects on stress, quality of life, and symptoms in persons with chronic fatigue syndrome.

OBJECTIVE: The present pilot study was designed to test the effects of a 12-week group-based cognitive behavioral stress management (CBSM) intervention on stress, quality of life, and symptoms in chronic fatigue syndrome (CFS). We hypothesized that participants randomized to CBSM would report improvements in perceived stress, mood, quality of life, and CFS symptomatology from pre- to postintervention compared to those receiving a psychoeducational (PE) seminar control. METHOD: We recruited 69 persons with a bona fide diagnosis of CFS and randomized 44 to CBSM and 25 to PE. Participants completed the Perceived Stress Scale (PSS), Profile of Mood States (POMS), Quality of Life Inventory (QOLI), and a Centers for Disease Control (CDC)-based CFS symptom checklist pre- and postintervention. RESULTS: Repeated measures analysis of variance revealed a significant Group×Time interaction for PSS, POMS-total mood disturbance (TMD), and QOLI scores, such that participants in CBSM evidenced greater improvements than those in PE. Participants in CBSM also reported decreases in severity of CFS symptoms vs. those in PE. CONCLUSIONS: Results suggest that CBSM is beneficial for managing distress, improving quality of life, and alleviating CFS symptom severity.