Proton transport through membranes induced by weak acids: A study of two substituted benzimidazoles

Summary We report here a kinetic study of the mechanism by which the weak acid TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) transports protons across phospholipid bilayer membranes. A previous kinetic study of the homologous dichloro compound, DTFB, revealed that the rate limiting step for proton translocation was the back diffusion of the neutral, HA, form of the weak acid; we conclude here that this is also the rate limiting step for proton translocation with TTFB. At high concentrations of either DTFB or TTFB the charged permeant species is an HA ₂ ^– complex. The kinetic analysis and independent measurements reveal that the permeability of the membrane to HA and adsorption coefficients of A^– and HA are an order of magnitude higher for TTFB than for DTFB. When either DTFB or TTFB was present in a solution where the pH was less than the pK of the weak acid, an unusual relaxation in the current was noted on application of a voltage step. The amplitude of the relaxation decreased as the voltage was increased. This relaxation is possibly due to a reorientation of the benzimidazole molecules at the membrane-solution interface. We also report experiments performed with DTFB on mitochondria. It was possible to reconcile these results with the bilayer data and, therefore, with the chemiosmotic hypothesis by postulating that the dielectric constant of the mitochondrial membrane is greater than that of a bilayer formed with decane as a solvent. To demonstrate the effect of dielectric constant on permeability, we replaced decane by 1-chlorodecane. This increased the capacitance of the artificial bilayer by a factor of two and the permeability of the bilayer to the A^– form of DTFB by two orders of magnitude.     

Regulation of RNA synthesis in yeast III

Summary Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse-long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.     

Perinatal methadone exposure and brain development: a biochemical study

Abstract— The neurochemical effect of maternally administered methadone (5 mg/kg, DL-methadone-HCI) on the brain (including the olfactory bulbs, cerebellum, and brain stem) and cerebellum of offspring exposed during gestation and/or lactation was studied in 10-, 21-, and 60-day old rats. Brain weights were significantly reduced in all methadone-exposed groups at 10 days of age, while only those rats subjected to methadone during gestation or lactation had deficits in brain weights at day 21; no differences were found at 60 days. Brain DNA content was significantly reduced in all opiate-exposed offspring at every age examined, but RNA/DNA and protein/DNA ratios were only consistently increased in rats of the gestation group. Cerebellar weight was reduced at 10 days in the gestation-lactation pups, at 21 days in rats of the gestation and lactation groups, and at 60 days in animals of the gestation and gestation-lactation groups. Cerebellar DNA content was significantly decreased in pups of the gestation group at every age investigated, but only reduced at 21 days in the lactation group and at 60 days in the gestation-lactation group. Rats in the lactation group had the greatest number of alterations in terms of RNA and protein, with the most noticeable being decreases in mean cellular RNA content on days 21 and 60 and a reduction in the mean cellular protein content on day 60. These data suggest that prenatal and/or postnatal methadone treatment affects the biochemical maturation of the central nervous system; deficits in neurons and/or glia, as well as a reduction in myelination, might be reflected in these changes.     

Partial purification and some properties of a neutral sulfhydryl and an acid proteinase from Entamoeba histolytica.

The partial purification of two intracellular proteinases from the protozoan parasite Entamoeba histolytica is reported. One of these enzymes is an acid proteinase exhibiting maximum activity at pH 3.5 (hemoglobin substrate), is little affected by a range of inhibitors or activators, and is presumed to be similar to cathepsin D. Also present is a neutral proteinase exhibiting optimum activity at pH 6.0 (azocasein) but only poorly hydrolyzing either hemoglobin or serum albumen. This latter enzyme displayed no metal ion requirement, but was markedly inhibited by thiol-blocking agents and activated by free sulhydryl-containing compounds.     

Robustness of G Proteins in Alzheimer''s Disease: An Immunoblot Study

Many of the neurotransmitter systems that are altered in senile dementia of the Alzheimer type are known to mediate their effects via G proteins, yet the integrity of guanine nucleotide-binding proteins (G proteins) in Alzheimer's diseased brains has received minimal investigation. The aim of this study was to establish whether the level of G alpha subunits of five G proteins was altered in Alzheimer's disease. We used immunoblotting (Western blotting) to compare the amounts of Gi1, Gi2, GsH (heavy molecular weight), GsL (light molecular weight), and Go in the frontal cortex and hippocampus, two regions severely affected by the disease, and the cerebellum, which is less severely affected. The number of senile plaques was also quantified. We report that there was no significant difference in the level of these G alpha subunits between Alzheimer's diseased and age-matched postmortem brains. These results suggest that alterations in the amount of G protein alpha subunits are not a feature of Alzheimer's disease.     

The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA.

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.     

Direct evidence for nitric oxide formation from glyceryl trinitrate during incubation with intact bovine pulmonary artery.

It has been proposed that the mechanism of the vasodilator action of glyceryl trinitrate (GTN) involves biotransformation to nitric oxide. A sensitive chemiluminescence method for nitric oxide determination was used to test this hypothesis. In four experiments, bovine pulmonary artery (BPA) was incubated with GTN (0.1 mM) in Krebs' solution (2 mL) containing 30 mM KCl, and in anaerobic conditions using 95% Ar - 5% CO2, in a sealed micro-Fernbach flask (6.2-mL volume). After incubation for 2, 5, 10, or 20 min at 37 degrees C, 400-microL aliquots of headspace gas were removed and injected into a redox chemiluminescence detector. Nitric oxide formation was first measurable at 5 min (76 +/- 53 pmol/g wet wt. BPA), and increased with incubation time (174 +/- 46 pmol/g wet wt. BPA after 10 min and 310 +/- 67 pmol/g wet wt. BPA after 20 min). This is the first direct chemical measurement of nitric oxide formation during interaction of GTN with vascular smooth muscle. These data support the concept that GTN is a nitrovasodilator prodrug acting via the formation of nitric oxide.