ABSTRACT
We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.
Subject(s)
Luminescent Measurements , beta-Galactosidase/analysis , Biotechnology , Escherichia coli/enzymology , Evaluation Studies as Topic , SpectrophotometryABSTRACT
During postnatal differentiation, epidermal growth factor (EGF) receptor is expressed by all major cell types of the cervix. Computer-assisted image analysis confirmed the highest concentration of EGF receptor is in the epithelial cells. Flow cytometric analysis of subpopulations of epithelial cells from estrous rabbits showed the mucous secreting cells had the highest concentration of EGF receptor, i.e. 1-1.5 x 10(5) receptors per cell. Because the mucous secreting cells are targets for steroid hormones it seemed likely that steroids regulate EGF receptor expression. To investigate this possibility, hormone-dependent changes in EGF receptor expression were quantified by flow cytometry. Ovariectomy and the treatment of ovariectomized animals with estradiol altered forward angle light scatter and side scatter signals which correlated with cell size and secretory granule content, respectively. However, the number of epithelial cells and the number of EGF receptors per cell were unaffected. Progesterone treatment of ovariectomized animals dramatically reduced the number of EGF receptors on the mucous secreting cells, accounting for a 43% reduction in the total EGF receptor content of the epithelium. The treatment of neonates with diethylstilbestrol did not change the number of EGF receptors in endocervical epithelial cells when examined in adulthood. However, the number of mucous secreting cells was decreased, thereby reducing the EGF receptor content of the epithelium 19-36% compared to estrous and estradiol-treated animals. These results provide the first evidence that progesterone regulates EGF receptor on mucous secreting cells in the endocervix and that diethylstilbestrol treatment alters the EGF receptor content of the epithelium by altering its cellular composition.
