No abstract

Protein kinase C-delta is activated during apoptosis, following proteolytic cleavage by caspase 3. Furthermore, overexpression of the catalytic kinase fragment of PKC-delta induces the nuclear phenotype associated with apoptosis, though the molecular basis of this effect has not been determined. In these studies we have examined the role of PKC-delta in the disassembly of the nuclear lamina at apoptosis. The nuclear lamina is disassembled during mitosis and apoptosis and mitotic disassembly involves hyperphosphorylation of lamin proteins by mitotic lamin kinases. During apoptosis, lamin proteins are degraded by caspase 6 and the contribution made by phosphorylation has not been proven. We show here that protein kinase C-delta co-localized with lamin B during apoptosis and activation of PKC-delta by caspase 3 was concomitant with lamin B phosphorylation and proteolysis. Inhibition of PKC-delta delayed lamin proteolysis, even in the presence of active caspase 6, whilst inhibitors of mitotic lamin kinases were without effect. In addition recombinant human PKC-delta was able to phosphorylate lamin B in vitro suggesting that its actions are direct and not via an intermediary kinase. We propose that PKC-delta is an apoptotic lamin kinase and that efficient lamina disassembly at apoptosis requires both lamin hyperphosphorylation and caspase mediated proteolysis.

IntroductionThis review is not intended as a summary of research and development of immunoassays and liquid chromatography-mass spectrometry but rather as a brief summary of the pros and cons of each technology applied to the quantitative measurement of biomolecules primarily in biofluids. The ability to detect and quantitate biomolecules revolutionised science in the twentieth century and continues to this day. It allows scientists, for example, to conduct research to find and validate biomarkers, but has found its most prominent role in diagnostic applications where it is used to monitor the presence and levels of biomolecules in human samples for the diagnosis and monitoring of disease, in food and beverages to ensure food safety and authenticity and in the environment to monitor the presence and levels of contaminants of ground, waste and drinking water. The growth in personalised or precision medicine will be accompanied by an increased need to detect and quantify panels of biomolecules as biomarkers, as well as a range of drugs taken as treatment or as a measure to delay or prevent onset of disease, following the paradigm of the right drug for the right patient at the right time and importantly at the right dose. Increased health and safety regulations demand more testing of food and beverage and environmental samples. With increasing need for biomolecule detection and quantitation comes the demand for high-throughput, lower cost per sample analysis and more accurate results. Traditionally, immunoassays have been the technology of choice for the detection and quantitation of biomolecules. However, over the past two decades alternative technologies have been shown to offer a complementary role to immunoassays. Liquid chromatography-mass spectrometry (LC-MS) is one such technology and for some applications has been shown to offer a powerful alternative to immunoassays. With the increasing demands for routine biomolecule quantitation coming from a broad range of fields, are immunoassays the best solution to keep pace with the increased throughput needed, demand for lower cost per sample and improved accuracy Immunoassays have been the technology of choice for the analysis of biomolecules for many decades across a wide range of applications in research, diagnostics and infectious disease monitoring. There are good reasons for the wide adoption of immunoassays but even such a well established and characterised technique has limitations and as such investigators are looking at alternative technologies. One such alternative is liquid chromatography (LC) and, more specifically, liquid chromatography coupled with mass spectrometry (LC-MS). This article will review both immunoassay and LC and LC-MS technologies and methodologies and discuss the advantages and limitations of both approaches. In addition, the next developments that will need to occur before there is widespread adoption of LC and LC-MS technology preferentially over immunoassays will be examined.