A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase

Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR–FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.     

A comparison of treatment modes in the management of myofascial pain dysfunction syndrome

This research compares different treatment regimes for the management of chronic facial pain associated with the masticatory musculature. Twenty-one females meeting specific criteria were randomly assigned to one of three treatment conditions: a dental splint and physiotherapy program; a relaxation program utilizing progressive muscle relaxation, biofeedback, and stress management techniques; or a minimal treatment program involving transcutaneous electrical nerve stimulation. Improvement was assessed through a dental examination, self-monitoring of pain, and an assessment of EMG activity during resting and task conditions. Significant changes were obtained in response to all treatment programs. The treatment programs differed only in the relative pattern of treatment effects obtained from the self-report monitoring of pain. The data are consistent with the concept of MPD as a psychological response to stress which maintains chronic pain through increased muscle tension in the jaw.     

Thermal behavior of synthetic sphingomyelin-cholesterol dispersions.

The thermotropic behavior of aqueous dispersions of palmitoylsphingomyelin-cholesterol and lignoceryl-sphingomyelin-cholesterol mixtures has been examined by high-sensitivity differential scanning calorimetry. When less than 25 mol % cholesterol is mixed with either sphingomyelin, the calorimetric endotherm is composed of a sharp and a broad component. The sharp-component enthalpy change decreases as the mole percent cholesterol increases with the extrapolated zero enthalpy point being 25 to 30 mol %. With palmitoylsphingomyelin, the temperature of maximum heat capacity of the sharp component decreases monotonically with increasing cholesterol content, while the lignocerylsphingomyelin sharp-component maximum remains constant until more than 20 mol % sterol is present. The broad-component enthalpy change maximizes at 3--4 kcal/mol between 10 and 20 mol % cholesterol and decreases as the ratio of cholesterol is increased or decreased from this range for both sphingomyelins. The results are compared with those from a previous study on dipalmitoylphosphatidylcholine-cholesterol mixtures and are interpreted as evidence for the coexistence of cholesterol-rich and cholesterol-poor phases.     

Human behavioral ecology: II

Human behavioral ecology is an interdisciplinary field of study applying theory from evolutionary ecology to a variety of anthropological questions. In Part I of this essay,¹ which appeared in an earlier issue, I surveyed key theoretical and methodological elements of the field, and summarized representative studies and issues in the topical area of subsistence strategies. In Part II, I turn to studies of reproductive strategies, and those analyzing patterns of cooperation and competition in an ecological and adaptive framework. I conclude with a brief look at possible future developments in the field.     

The effectiveness of sampling methods in detecting copulatory behavior in Macaca arctoides

The copulatory behavior of a social group of 18 stumptail macaques was observed for 171 hours over a period of 5 months. The occurrence of copulation and several quantitative measures of copulation were compared using sampling periods of 2 hours daily and continuous dawn-to-dusk observation. In this environment copulation was found to occur in brief bouts of relatively short duration. Copulation was detected on 34% of the days observed with 2 hr sampling and 100% of the days with DTD sampling. Thus, brief limited-time samples of behavior are not adequate for reliably detecting copulation in this species under social group-living conditions.     

The benefits of costly signaling: Meriam turtle hunters

Hunting, particularly when it involves large game that is extensivelyshared, has been suggested to serve as a form of costly signalingby hunters, serving to attract mates and allies or to detercompetitors. Empirical evidence presented elsewhere on turtlehunting practiced by Meriam people of Torres Strait, Australia,supports several key predictions of the costly signaling account.Here we present evidence from the same study bearing on anotherkey prediction, that signalers (hunters) gain social and reproductivebenefits. Specifically, we find that successful hunters gainsocial recognition, have an earlier onset of reproduction, achievehigher age-specific reproductive success, and gain higher qualitymates, who also achieve above-average reproductive success.Meriam hunters also average more mates (women who bear theiroffspring) and more co-resident sexual partners than other men,and these partners (but not mates) are significantly younger.Several lines of evidence thus support the idea that huntingis a form of costly signaling in this population. Alternativehypotheses involving reciprocity (from grateful recipients ofmeat) and direct offspring provisioning by hunters are not consistentwith available evidence, but in the absence of experimentalmanipulation we cannot rule out a role for phenotypic correlation.