An equilibrium between distorted and undistorted DNA in the adult chicken beta A-globin gene

We have used single strand specific nucleases to map DNA distortion in the adult chicken beta A-globin gene. We have detected two structures of that kind and have mapped nuclease-cutting sites at one base resolution. One prominent site is centered at -190 relative to the RNA capping site and is positioned at the center of a stretch of contiguous C residues. The second site is near the first intron/exon junction (+620) and appears as a series of discrete 1-base-long enzyme-cutting sites. Based upon the pattern of nuclease cutting and the kinetics of nuclease cutting we conclude that the "poly(C)" stretch may assume a looped geometry in supertwisted DNA molecules which is similar to that proposed by Felsenfeld (Nickol, J. M., and Felsenfeld, G. (1983) Cell 35, 467-477). We show that S1 nuclease cuts within the intron occur mainly at the end points of polypurine segments and suggest that such end points may assume a distorted transitional geometry. We find that Neurospora crassa endonuclease cuts both the promotor and intron sites in linear DNA molecules but that in linear DNA the cutting process is limited by a first order conformation change of the DNA substrate. Based upon those kinetics we propose that in unstressed DNA, each of the two sites can convert between a distorted and undistorted geometry. In the enzyme assay buffer at 37 degrees C, the time constant for the equilibrium is nearly 10 h for the promotor site and 7 h for the intron.     

Modulation induction of the T3 antigen by OKT3 antibody is monocyte dependent

We investigated the influence of monocytes on the susceptibility of the T3 antigen on human T cells to modulation induction by OKT3 antibody. In the absence of monocytes, the T3 antigen was only minimally susceptible to modulation. After the addition of 20% monocytes to the culture, however, complete modulation was readily observed. Furthermore, we found that even in the absence of OKT3 antibody, monocytes were able to down-regulate the expression of the T3 antigen, although to a lesser extent. The ability of monocytes to enhance antigenic modulation proved to be a more general phenomenon. Each individual T cell antigen, however, differed in its susceptibility to modulation by antibody, monocytes, or both, thereby establishing its own characteristic pattern. In addition, after complete modulation of the T3 antigen, the addition of monocytes to the culture thereafter had a distinct inhibitory effect on the reexpression of the T3 antigen. Monocyte enhancement of T3 modulation is significantly reduced when using the OKT3 F(ab')2 fragment, as is OKT3 mitogenesis. After pulsing the monocytes with OKT3 antibody before adding them to the culture, T3 modulation became nearly complete even in the absence of added OKT3 antibody. Monocyte-induced modulation proved not to be MHC restricted, thus allowing for comparative analysis of this effect between monocytes and other cell types. A moderate, however, incomplete modulation enhancement was observed with the human monocyte cell line U937 and with Daudi cells. This finding proved to coincide with the distinct ability of these cell lines to bind OKT3 antibody by their Fc receptors, as was the case with monocytes. In contrast, neither Fc receptor binding nor T3 modulation enhancement was observed with the cell lines Cess and G7. In addition, no effective T3 modulation was observed with glutaraldehyde-fixed monocytes. The overall results seem to indicate that effective modulation of the T3 antigen by OKT3 antibody requires the active participation of Fc receptors on monocytes.     

The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

Application of macroscopic balances to the identification of gross measurement errors

A systematic method is presented which is capable of both detecting the presence of grossly biased measurement errors and locating the source of these errors in a bioreactor through statistical hypothesis testing. Equality constraints derived from material and energy balances are employed for the detection of data inconsistencies and for the subsequent identification of the suspect measurements by a process of data analysis and rectification. Maximum likelihood techniques are applied to the estimation of the states and parameters of the bioreactor after the suspect measurements have been eliminated. The level of significance is specified by the experimenter while the measurments are assumed to be randomly, normally distributed with zero mean and known variances. Two different approaches of data analysis, batchwise and sequential, that lead to a consistent set of adjustments on the experimental values, are discussed. Several examples based on the fermentation data taken from literature sources are presented to demonstrate the utility of the proposed method, and one set of data is solved numerically to illustrate the computational aspect of the algorithm.