Electrical potential difference, sodium absorption and potassium secretion by the human rectum during carbenoxolone therapy.
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The transmucosal electrical potential difference (pd) and the sodium and potassium net fluxes were measured in the rectum of subjects taking carbenoxolone. There was a rise in transmucosal pd persisting throughout treatment in all subjects which was accompanied by an increase in sodium absorption and potassium secretion. Comparison of the pd changes produced by carbenoxolone with those due to the mineralocorticoid 9-alpha-fluorocortisol showed that carbenoxolone had about 1/1000th the potency on a weight basis and the two drugs appeared to be additive in their effects. Topical instillation of carbenoxolone into the rectum produced an elevation of pd which persisted for three days. Amiloride and bendrofluazide did not interfere with these actions of carbenoxolone but spironolactone abolished them. One patient who developed fluid retention and hypokalaemia had a rectal pd similar to that of the other patients who had no side effects. (+info)
Method for the measurement of antioxidant activity in human fluids.
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AIM: To develop a new, simple, and cheap method for estimating antioxidant activity in human fluids. METHODS: The assay measured the capacity of the biological fluids to inhibit the production of thiobarbituric acid reactive substances (TBARS) from sodium benzoate under the influence of the free oxygen radicals derived from Fenton's reaction. A solution of 1 mmol/litre uric acid was used as standard. RESULTS: The following mean (SD) antioxidative activities were found (as uric acid) in the various biological fluids: serum, 2.04 (0.20) mmol/litre; urine, 176.5 (25.6) micromol/litre; cerebrospinal fluid, 95.0 (26.9) micromol/litre; aqueous humour oculi, 61.25 (9.9) micromol/litre; saliva, 838.5 (48.2) micromol/litre; tears, 247.0 (17.0) micromol/litre; ascites fluid, 270.0 (63.3) micromol/litre; kidney cyst fluid, 387.1 (28.1) micromol/litre. Small samples of the biological material were needed for the analyses: 10 microl of serum and 50-100 microl of other body fluids. In the sera of 48 healthy individuals there was a significant positive correlation between values obtained with the Randox method (as a reference method) and the new method proposed here (correlation coefficient, 0.8728; mean difference between methods, <0.4%). CONCLUSIONS: This method is easy, rapid, reliable, and practical for the routine measurement of total antioxidant activity in serum and other human body fluids. Small samples of biological material are needed for the analyses and the results are comparable with the reference (Randox) method. (+info)
Mutational analysis of conserved residues in the GCN5 family of histone acetyltransferases.
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GCN5 is a critical transcriptional co-activator and is the defining member of a large superfamily of N-acetyltransferases. GCN5 catalyzes the transfer of an acetyl group from acetyl-CoA to the epsilon-amino of lysine 14 within the core H3 histone protein. Previous biochemical analyses have indicated a fully ordered kinetic mechanism. Recent structural studies have implicated several conserved residues in catalysis and substrate binding. Here the roles of Glu-173, His-145, and Asp-214 in yeast GCN5 have been evaluated using site-directed mutagenesis, steady state and pre-steady state kinetics, pH analysis, isotope partitioning, and equilibrium binding studies. The results with wild type and E173Q, H145A, and D214A mutants are consistent with chemical catalysis being rate-determining in turnover. All mutants exhibited K(d) values (3.5-8.5 microm) for AcCoA that were similar to wild type enzyme, indicating no functional role for these residues in AcCoA binding. The E173Q mutant demonstrated a approximately 500-600-fold decreases in k(cat) and k(cat)/K(m),(H3), consistent with Glu-173 acting as the general base catalyst as proposed previously. No significant effect was observed on substrate binding steps. His-145 was identified as a residue in the peptide binding cleft that must be unprotonated (pK(a) = 5.8) for peptide binding and likely hydrogen-bonds to the Ser-10 hydroxyl of histone H3. His-145 also contributes to lowering the pK(a) value (by 0.8 units) of general base Glu-173 through a water-mediated hydrogen bond to the carboxylate side chain. Analysis of D214A revealed an obligate protein isomerization step that occurs after AcCoA binding and permits efficient peptide binding. Asp-214 is part of a conformationally flexible loop that mediates the isomerization by stabilizing distinct conformers of the protein. (+info)
Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase.
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The interaction of L-tyrosine, L-tyrosyladenylate and tRNA-Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a K-d of 2 x 10-5 M at pH 7.8 and 23 degrees C. The tyrosyl-tRNA synthetase--tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNA-Tyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having K-d values of 2 x 10-7 M and 1.3 x 10-6 M respectively at pH 6.5 and 25 degrees C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme. (+info)
Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.
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Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles. (+info)
Partial isolation of a pheromone accelerating puberty in female mice.
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The sexual development of female mice is accelerated by exposure to an adult male or to male urine. The component of the urine responsible for this effect is androgen-dependent, heat labile, nondialysable, precipitatable with ammonium sulphate, and is not extractable in ether. These results indicate that the pheromone causing accelerated sexual development is associated with a protein component of male urine. Tests of the active fraction after digestion with proteolytic enzymes suggest that the pheromone may be a portion of a protein or a substance bound to a protein. (+info)
Genistein, a soy isoflavone, is a potent alpha-glucosidase inhibitor.
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Genistein is an isoflavone that is known to be contained in soybean. It was proved that genistein plays a pivotal role in homeostasis in the human body. In the course of screening for useful alpha-glucosidase inhibitors, we isolated and identified genistein as a candidate for alpha-glucosidase inhibitor from fermentation broths of a Streptomyces sp. Genistein was shown to be a reversible, slow-binding, non-competitive inhibitor of yeast alpha-glucosidase with a K(i) value of 5.7x10(-8) M when the enzyme mixture was pretreated with genistein. These results show a possibility that genistein could be a useful tool for metabolic disorders. (+info)
A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media.
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Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule. (+info)