Retention of taste quality following brief exposure to sucrose

Rats were given either 32% sucrose or 4% sucrose during preshiftand 4% sucrose postshift. The preshift and postshift sessionseach consisted of single five minute access periods to the sucrosesolutions. Measures of lick-rate showed that the shifted ratslicked less of the 4% sucrose than the unshifted rats, a negativecontrast effect, and that this negative contrast effect occurredwith intervals ranging from 1 hr to 70 hrs separating the briefpreshift and postshift exposure periods. The results were discussedin terms of the duration of gustatory memories, particularlyin reference to the conditioned aversion literature and somerunway contrast phenomena.     

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.     

Measurement and characterization of postischemic free radical generation in the isolated perfused heart

Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the postischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g parallel = 2.033 and g perpendicular = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and aN = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Competitive Fitness of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed Cultures with and without Food in the U.S. Food and Drug Administration Enrichment Protocol

Thirteen different serotypes of the food-borne pathogen Listeria monocytogenes have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence L. monocytogenes is described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM). We compared three strains of L. monocytogenes serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate Listeria species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.     

The Effect of Coumarin on Growth, Production of Dry Matter, Protein and Nucleic Acids in Roots of Maize and Wheat and the Interaction of Coumarin with Metabolic Inhibitors

Effects of coumarin on fresh weight, dry matter, protein and nucleic acid content per cell in attached roots of maize and wheat and in whole excised elongation zones of maize were determined. The inhibition in cell length exerted by coumarin did not correspond to an inhibition of the net synthetic capacity. Coumarin treatment increased the cell surface, the production of dry matter and the protein content per cell. The dry matter and the protein content per unit surface was slightly increased or unaffected. The effect of coumann on cell shape seemed to be independent of that on dry matter production and net protein synthesis. The same was found in excised elongation zones. —The net DNA-synthesis per cell was slightly increased in attached roots by coumann treatment, but this effect was probably not correlated with the morphogenetic changes. Inhibition of DNA-synthesis with hydroxyurea did not alter the coumarin induced changes in cell shape. —The net RNA-synthesis per cell was slightly decreased after coumarin treatment, but the net RNA-synthesis per cell and the morphogenetic effects exerted by coumarin were not related with each other. Inhibition of m-RNA-synthesis with actinomycin D did not prevent the effects of coumarin on cell division, cell expansion, dry matter production and net protein synthesis. The same was true for inhibitors of protein synthesis, puromycin and p-fluorophenyl-alanine. The findings are in support of the view that coumarin affects already existing structures or enzymes. —Comparisons between coumarin and the uncouplers, DNP and dicoumarol, showed that the effects of coumarin were not, solely, due to uncoupling. SH-protecting agents, BAL, DTE and glutathione, did, with few exceptions, not reduce the morphogenetic effects of coumarin.