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Chronic ethanol consumption is associated with increased protein oxidation and decreased proteolysis in the liver. We tested the hypothesis that even single-dose treatment with ethanol or bromotrichloromethane causes increased protein oxidation and a distinct proteolytic response in cultured hepatocytes. HepG2 cells were treated for 30 min with ethanol, H(2)O(2) and bromotrichloromethane at various nontoxic concentrations. Protein degradation was measured in living cells using [35S]-methionine labeling. Protein oxidation, and 20S proteasome activity were measured in cell lysates. Oxidized proteins increased immediately after ethanol, H(2)O(2), and bromotrichloromethane exposure, but a further significant increase 24-h after exposure was observed only following ethanol and bromotrichloromethane treatment. All three reagents caused a significant increase of the overall intracellular proteolysis at rather low concentrations, which could be suppressed by the proteasome inhibitor lactacystin. A decline of proteolysis observed at higher-subtoxic-concentrations was not related to decreased proteasome activity. Preincubation with ketoconazole or 4-methylpyrazole completely prevented the ethanol- and bromotrichloromethane-induced but not the H(2)O(2)-induced protein oxidation and proteolysis, suggesting strongly an enzyme-mediated generation of reactive oxygen species. In conclusion single-dose exposure with ethanol or haloalkanes causes increased protein oxidation followed by an increased proteasome-dependent protein degradation in human liver cells.

During cold exposure, animals upregulate their metabolism and food intake, potentially exposing them to elevated reactive oxygen species (ROS) production and oxidative damage. We investigated whether acute cold (7 +/- 3 degrees C) exposure (1, 10, or 100 h duration) affected protein oxidation and proteasome activity, when compared to warm controls (22 +/- 3 degrees C), in a small mammal model, the short-tailed field vole Microtus agrestis. Protein carbonyls and the chymotrypsin-like proteasome activity were measured in plasma, heart, liver, kidney, small intestine (duodenum), skeletal muscle (gastrocnemius), and brown adipose tissue (BAT). Trypsin-like and peptidyl-glutamyl-like proteasome activities were determined in BAT, liver, and skeletal muscle. Resting metabolic rate increased significantly with duration of cold exposure. In skeletal muscle (SM) and liver, protein carbonyl levels also increased with duration of cold exposure, but this pattern was not repeated in BAT where protein carbonyls were not significantly elevated. Chymotrpsin-like proteasome activity did not differ significantly in any tissue. However, trypsin-like activity in SM and peptidyl-glutamyl-like activity in both skeletal muscle and liver, were reduced during the early phase of cold exposure (1-10 h), correlated with the increased carbonyl levels in these tissues. In contrast there was no reduction in proteasome activity in BAT during the early phase of cold exposure and peptidyl-glutamyl-like activity was significantly increased, correlated with the lack of accumulation of protein carbonyls in this tissue. The upregulation of proteasome activity in BAT may protect this tissue from accumulated oxidative damage to proteins. This protection may be a very important factor in sustaining uncoupled respiration, which underpins nonshivering thermogenesis at cold temperatures.

Flow cytometry has become a widely-used and powerful tool for the characterization of cells according to their expression of specific proteins. However, sensitivity of this method is still limited since conventionally labeled antibodies can be conjugated with at maximum 1-10 dye molecules. This fact resulted in the need to develop new techniques in order to identify molecules which are expressed in very low but functionally relevant amounts. In the past, we have successfully used a liposome-based high-sensitivity immunofluorescence technique to measure the expression of low abundant membrane bound glucocorticoid receptors (mGR) on different cell types. The use of this technique allows the detection of as few as 50-100 antigen molecules per cell which is due to a 100-fold to 1000-fold increase in fluorescence signal intensity compared with conventional methods. The higher sensitivity is achieved since thousands of dye molecules can be enclosed in liposomes. Another modern high-sensitivity immunofluorescence staining method is the purchasable Fluorescence Amplification by Sequential Employment of Reagents (FASER) procedure. Here, we aimed at comparing sensitivity and specificity of these two techniques for the detection of the mGR. Our data demonstrate the FASER technique to be more sensitive and also more specific for the detection of mGR as compared to the liposome technique. However, both methods have advantages and disadvantages which are discussed in detail. [ABSTRACT FROM AUTHOR]

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H2O2 induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation. Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin. [ABSTRACT FROM AUTHOR]