Over the last few years, an increased awarenes of endocrine disrupting chemicals (EDCs)

Fluorescence polarization (FP) equilibrium binding assays differ from other types of binding studies in one important regard: they require no steps to separate free from bound tracer and are therefore fast, simple and accurate.

Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wide range of environmental and industrial chemicals. While it is clear that in vivo methods will be required to identify adverse effects produced by these chemicals, in vitro assays can define particular mechanisms of action and have the potential to be employed as rapid and low-cost screens for use in large scale EDC screening programs. Traditional estrogen receptor (ER) binding assays are useful for characterizing a chemical's potential to be an estrogen-acting EDC, but they involve displacement of a radioactive ligand from crude receptor preparations at low temperatures. The usefulness of these assays for realistically determining the ER binding interactions of weakly estrogenic environmental and industrial compounds that have low aqueous solubility is unclear. In this report, we present a novel fluorescence polarization (FP) method that measures the capacity of a competitor chemical to displace a high affinity fluorescent ligand from purified, recombinant human ER-[alpha] at room temperature. The ER-[alpha] binding interactions generated for 15 natural and synthetic compounds were found to be similar to those determined with traditional receptor binding assays. We also discuss the potential to employ this FP technology to binding studies involving ER-ss and other receptors. Thus, the assay introduced in this study is a nonradioactive receptor binding method that shows promise as a high throughput screening method for large-scale testing of environmental and industrial chemicals for ER binding interactions.ImagesFigure 2Figure 3Figure 4

Abstract. Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.

A strain of pigs bearing three immunogenetically defined lipoprotein-associated markers (allotypes), designated Lpb5, Lpr1, and Lpu1, has marked hypercholesterolemia on a low fat, cholesterol-free diet. Unlike individuals with familial hypercholesterolemia or WHHL rabbits, the affected pigs have normal low density lipoprotein receptor activity. The animals, by 7 months of age, have extensive atherosclerotic lesions in all three coronary arteries. This strain of pig represents an animal model for atherosclerosis and hypercholesterolemia associated with mutations affecting the structures of plasma lipoproteins. One of the variant apolipoproteins, Lpb5, is apolipoprotein-B. A second variant apolipoprotein (Lpr1), termed apo-R, is a 23-kilodalton protein present in both the very low density (d less than 1.006 g/ml) and the very high density (d greater than 1.21 g/ml) fractions of pig plasma. Isoforms of this protein correlate with two Lpr alleles, Lpr1 and Lpr2. The Lpr genes segregate independently of the Lpb5 and Lpu1 alleles. The Lpu1 allotype is a component of low density lipoprotein and is genetically linked to Lpb5.