Plr Bonner

Electrospray mass spectrometry for the identification of MHC class I-associated peptides expressed on cancer cells

Electrospray ionisation (ESI) mass spectrometry (MS) has been used extensively for the detection of peptides presented by major histocompatibility complex (MHC) molecules. This review focuses on the optimisation of electrospray mass spectrometry and the use of tandem mass spectrometry to sequence MHC class I peptides. We review the isolation of MHC class I peptides from the surface of cells with particular reference to tumour cells. In addition, we also discuss the advantages and disadvantages of the methods available to concentrate and fractionate the peptides prior to analysis by electrospray mass spectrometry.

Effects of phenyl saligenin phosphate on cell viability and transglutaminase activity in N2a neuroblastoma and HepG2 hepatoma cell lines

The main aim of this study was to determine whether sub-lethal concentrations of the organophosphate compound phenyl saligenin phosphate (PSP) could disrupt the activity of the Ca(2+)-activated enzyme tissue transglutaminase (TGase 2) from cultured cell lines of neuronal (N2a) and hepatic (HepG2) origin. The results indicated that PSP added directly to cytosol extracts from healthy cells was able to inhibit TGase 2 activity by 40-60% of control levels at sub-lethal concentrations (0.1 microM) that were approximately 100-fold lower than their IC(50) values in cytotoxicity assays. Following 24h exposure of N2a cells to 0.3 and 3 microM PSP in situ, a similar reduction in activity was observed in subsequent assays of TGase 2 activity. However, significantly increased activity was observed following in situ exposure of HepG2 cells to PSP (ca. 4-fold at 3 microM). Western blotting analysis indicated slightly reduced levels of TGase 2 in N2a cells compared to the control, whereas an increase was observed in the level of TGase 2 in HepG2 cells. We suggest that TGase 2 represents a potential target of organophosphate toxicity and that its response may vary in different cellular environments, possibly affected by its expression pattern.

Effects of chlorpyrifos on transglutaminase activity in differentiating rat C6 glioma cells.

The organophosphorothioate compound chlorpyrifos (CPF) is a widely used pesticide, which is known to inhibit the differentiation of mouse N2a neuroblastoma and rat C6 glioma cells. This study in focused on the possible effects of CPF in the activity and expression of tissue transglutaminase (TGase 2) in differentiating C6 cells. Cells exposed for 24 h to 10 μM CPF, which had no effect on cell viability, exhibited a significant increase in cytosolic TGase 2 activity. Western blotting analysis indicated that there was no change in the cytosolic TGase 2 protein levels, suggesting that the enzyme was activated under these conditions. When commercially available TGase 2 was incubated with CPF in vitro, an increase in activity was also observed, suggesting that CPF might interact directly with TGase 2.