Flash spectroscopy of purple membrane.

Flash spectroscopy data were obtained for purple membrane fragments at pH 5, 7, and 9 for seven temperatures from 5 degrees to 35 degrees C, at the magic angle for actinic versus measuring beam polarizations, at fifteen wavelengths from 380 to 700 nm, and for about five decades of time from 1 microsecond to completion of the photocycle. Signal-to-noise ratios are as high as 500. Systematic errors involving beam geometries, light scattering, absorption flattening, photoselection, temperature fluctuations, partial dark adaptation of the sample, unwanted actinic effects, and cooperativity were eliminated, compensated for, or are shown to be irrelevant for the conclusions. Using nonlinear least squares techniques, all data at one temperature and one pH were fitted to sums of exponential decays, which is the form required if the system obeys conventional first-order kinetics. The rate constants obtained have well behaved Arrhenius plots. Analysis of the residual errors of the fitting shows that seven exponentials are required to fit the data to the accuracy of the noise level.     

Subnuclear localization and antitransforming activity of N-myc:beta-galactosidase fusion proteins.

N-myc expression is under stage- and tissue-specific regulation in mammalian development, but its function is totally unknown. We sought agents to block N-myc activity in order to infer from the effect the possible function of N-myc in the apparently complex processes. As candidates for such agents, we tested fusion genes encoding N-myc:beta-galactosidase fusion proteins for their effects on the formation of transformed foci of rat embryo primary fibroblasts as the result of transfection with N-myc and activated H-ras. One of the gene constructs very efficiently antagonized N-myc activity, as assessed by its effect on focus formation, but did not appreciably affect cell viability. The product of this gene was not only targeted to the nucleus but also accumulated in subnuclear loci which may represent the sites where normal N-myc proteins reside. The occurrence of antagonistic effect at a low stoichiometric ratio suggested that the fusion protein gene competed with the N-myc gene in a fashion analogous to a dominant negative mutation.     

Isolation of Temperature-Sensitive Membrane Mutants of Escherichia coli K-12

Mutants with impaired biosynthesis of unsaturated fatty acids or altered metabolism of the phospholipids were isolated at a rather high frequency from a set of temperature-sensitive lysis mutants. It is suggested that preselection for the lysis phenotype makes it possible to isolate several kinds of mutants affected in the integrity of the cytoplasmic membrane.     

An ELISA for detection of apoptosis.

We describe a simple and convenient enzyme-linked immunosorbent assay (ELISA) for the detection of apoptosis in tissue culture. An early event in apoptosis is DNA fragmentation followed by release of nucleosomes into the cytoplasm. Our sandwich assay uses a pair of monoclonal antibodies specific for two nucleosomal epitopes to capture and detect cytoplasmic nucleosomes onto the ELISA plate. Our assay is about 500 times more sensitive than the detection of apoptotic DNA ladder by agarose electrophoresis and is especially suited for the testing of large numbers of samples.