No abstract

Background and Aims Simple methods for determining Cu forms in wine do not currently exist. This study demonstrates the relevance of measuring a specific fraction of Cu in wine, and introduces simplified methods for its measurement utilising either colorimetry, or filtration in combination with routine measures of total Cu in wine. Methods and Results Electrochemically detectable Cu provided a threshold level of Cu that related directly to the accumulation of free hydrogen sulfide and free methanethiol in bottled wines. A simple colorimetric method using bicinchoninic acid was optimised to allow a rapid approximation of this fraction of Cu in white wine. By modification of the measurement conditions, an extra fraction of Cu associated with the partial release of Cu from thiols could also be quantified. The use of diatomaceous earth depth filters to isolate these fractions of Cu was also assessed. After passing red wine through the filters, the concentration of Cu in the filtrate correlated well with the electrochemically detectable Cu. Alternatively, for white wine the concentration of filtrate Cu overestimated this fraction of Cu and this was attributed to its inability to distinguish sulfhydryl‐free Cu from Cu loosely bound to thiols. Conclusions Multiple and simplified approaches have been devised to allow measurement of Cu fractions in wine. Significance of the Study These methodologies will allow an important fraction of Cu in wine to be measured with a spectrophotometer.

We examined what effects are exerted by expression of the bcl-2 gene and by treatment with nerve growth factor (NGF) on the intensity of apoptosis in cultured pheochromocytoma cells (PC12 cells). Half of these cells were transduced with the bcl-2 gene using lentiviral plasmids, and the respective two groups were denoted as bcl-2-PC12 and control (c) PC12. Then the cells were incubated in a serum-free medium in six different modes. One group of c-PC12 cells was incubated in this medium with no additional agents added, another group was incubated with 1.0 mM H 2 O 2 , and the third group was incubated with both 1 mM H 2 O 2 and 20 ng/ml NGF (groups 1-3). Cells of the another triad were incubated under the same conditions, respectively, but these were bcl-2-PC12 cells (groups 4-6). The apoptosis rate in each group after 1-h-long incubation was measured using a flow cytometry method. A bicinchoninic acid (BCA) technique was used for estimation of expression of Bcl-2 protein in the cultures. As was observed, the action of H 2 O 2 significantly increased the apoptosis rate in both c-PC12 and bcl-2-PC12 samplings, while simultaneous action of NGF considerably attenuated such increases. At the same time, values of the apoptosis rate for bcl-2-PC12 cells were much smaller than the respective values for c-PC12 cells under all the three modes of incubation. In H 2 O 2-treated cultures, the amount of Bcl-2 protein dropped, while the treatment with NGF counteracted such shifts. The content of this protein in the bcl-2-PC12 groups was much higher than in the c-PC12 groups. Thus, transduction with the bcl-2 gene significantly inhibits apoptosis in cultured PC12 cells, and a combined influence of expression of this gene and treatment with NGF produces a synergistic effect.