Acute effects of digitalis and enalapril on the neurohormonal profile of chagasic patients with severe congestive heart failure.

Chagasic patients with congestive heart failure are usually treated with digitalis and converting enzyme inhibitors. According to the neurogenic and dysautonomic theories, chagasic patients would not benefit from these drugs. To clarify this controversial issue, we have studied patients with congestive heart failure and suspected Chagas' heart disease. All patients received intravenous methyl-digoxin for 24 h and oral enalapril for 96 h. Blood samples for plasma norepinephrine, aldosterone and renin were taken at baseline, after acute digitalization and following enalapril. Based on the serology for Chagas' disease, the patients were divided into non-chagasic and chagasic patients. In the chagasic group three patients were in functional class III and 3 were in functional class IV. In the non-chagasic group five patients were in functional class III and 2 were in functional class IV. Both groups had a marked and quantitatively similar degree of neurohormonal activation. All patients improved at least one functional class and lost more than 5 kg of body weight with treatment. The chagasic patients had a statistically significant reduction in plasma norepinephrine (2262 +/- 1407 to 865 +/- 390, P < 0.008, pg/ml, M +/- S.D.), plasma aldosterone (330 +/- 168 to 155 +/- 75, P < 0.01, pg/ml, M +/- S.D.) and plasma renin activity (14 +/- 13 to 2 +/- 1.6 ng/ml per h, M +/- S.D., P < 0.05), with digitalis. Following enalapril, norepinephrine and aldosterone there was a further but non-significant reduction, when compared to postdigitalis values. These results indicated that chagasic patients do benefit from digitalis and enalapril. Furthermore, the prominent and significant reduction in all three neurohormones suggest that the parasympathetic and sympathetic systems of these chagasic and non-chagasic patients, are responding to the neuromodulatory effects of digitalis and enalapril.

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator.

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.

Lac repressor-operator interaction: DNA length dependence.

The interaction of the E. coli lac operon repressor with its operator DNA has been directly examined as a function of the length of operator-containing DNA. The apparent bimolecular association rate constants were calculated as ka = (kd/KD), where the dissociation equilibrium constant, KD and the dissociation rate constant, kd, were measured by nitrocellulose filter adsorption assays. The values obtained for the overall association rate constants are compared with theoretical association rate curves for specific mechanisms. Association of the repressor with short operator containing DNA fragments (less than 70 base pairs) occurs at rates expected of three-dimensional diffusion. Our data also imply that at longer DNA lengths a combination of three-dimensional diffusion with one-dimensional sliding along with hopping and/or intersegment transfer must be involved to facilitate the repressor operator association.