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Abstract

AbstractTea and crumpets, calpain and CRMP‐1‐CRMP‐4pp. 3712–3728If you order tea and crumpets, you expect a choice of accompanying nibbles and a fork to eat with. If a neuronal cell development program orders apoptosis due to low potassium, it expects proteins to cleave and proteases to do it with. In this case, the principal proteases have been identified as caspase‐3 and calpain, both cysteine proteases. The calpain targets include the collapsin response mediator proteins CRMP‐1 through CRMP‐4. Studying rat cerebellar granule cells, Liu et al. found that certain peptides could selectively inhibit either caspase‐3 or calpain, but both proteases had to be inhibited to stop apoptosis. More specifically, calpain was shown to promote apoptosis by clipping the carboxy termini of CRMP‐1, ‐2, and ‐4, and the amino terminus of CRMP‐3, although only clipped CRMP‐3 and ‐4 induced apoptosis. The techniques and tools used included 2‐D DIGE, MALDI‐TOF/TOF MS, Western blots, cloning and expression, and apoptosis assays. magnified imageTesting a heavyweight contenderpp. 3752–3764Professional boxers are made, not born. Vitamin D3 has been recognized as a potential heavyweight immunomodulator for several years but has proven “difficult to tame” due to its toxicity at effective in vivo dosage levels. Working with dendritic cells in culture and a modified form of vitamin D3, TX527, Ferreira et al. used proteomic tools and techniques, systems biology, and functional studies to examine the effects of TX527 on mature and immature dendritic cells. They evaluated differential protein profiles, protein interactome networks, and cell functions to show that ultimately TX527 initiates a process that yielded a more tolerogenic dendritic cell phenotype. Tools and techniques used included 2‐D DIGE, MALDI‐TOF/TOF MS, Western blots, network identification and analysis software, and functional assays. magnified imageO, that this too, too solid flesh would melt. Thaw, and resolve itself into a dew!pp. 3765–3771Hamlet bemoaning the difficulties of sample prep., ca. 1600‐1620. The quality of the input has a major impact on the outcome of an experiment, even with the best mass spec. technique. One common high‐resolution method of purifying proteins is preparative polyacrylamide gel electrophoresis. The drawback of PAGE is the difficulty extracting the separated proteins from the gel in reasonable yield. In‐gel digestion with trypsin reduces the sample to peptides that, mostly, elute from a crushed gel slice or spot. Kim et al. approached the problem from the other direction, developing a gel crosslinker that could be efficiently dissolved by mild acid, pH 3 to pH 5. Proteins were freed mostly intact and reasonably active. The hydrolyzed polyacrylamide could be removed by acetonitrile precipitation or ion exchange chromatography. Tools and techniques used: native 1‐D, 2‐D PAGE, ELISA, immunoaffinity assays. magnified image