Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications.

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a tag into proteins for relative quantitation by mass spectrometry. SILAC is a simple, yet powerful, method for investigating the dynamics of protein abundance and posttranslational modifications. Here, we provide detailed instructions for using this method to study protein complexes, protein-protein interactions, and the dynamics of protein abundance and posttranslational modifications. We expect that SILAC will become a routine technique because of its applicability to most areas of cell biology. We have also developed a Web site (http://www.silac.org) to provide researchers with updated information about this method and related resources.

High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay.

Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2-3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring approximately 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 microg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 microg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 microg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring <1 mg test material, giving a substantial increase in productivity. Any material selected for further development and inclusion in a formulation may require further confirmatory testing, e.g. using a human skin model assay for phototoxicity.

Comparative cytotoxicity of deoxynivalenol, nivalenol, their acetylated derivatives and de-epoxy metabolites.

The cytotoxicity of the de-epoxy metabolites of trichothecenes nivalenol (NIV) and deoxynivalenol (DON) was determined and compared with the cytotoxicity of the respective toxin with an intact epoxy group and their acetylated derivatives. The cytotoxic effects was determined by using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay assessing DNA-synthesis. The toxicity of NIV and DON expressed as the concentration inhibiting 50% of the DNA synthesis (IC(50)), was occurring at similar micromolar concentrations (1.19+/-0.06 and 1.50+/-0.34 microM). The toxicity of fusarenon X (4-acetyl NIV) in the assay was similar to the toxicity of NIV, and the toxicity of 15-AcDON was equal to the toxicity of DON. 3-AcDON was less toxic than DON and 15-AcDON. The IC(50) value for de-epoxy DON was 54 times higher in the assay than the IC(50) for DON, while the IC(50) of de-epoxy NIV was 55 times higher than the IC(50) for NIV. The results verify previous findings that the de-epoxidation is a detoxification reaction.

Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q).