Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved

Marino da Caramanico: giudice, giurista, intellettuale

Marino da Caramanico (1240? – 1285/7?) was without any doubt an important figure in the Angevin court. Nevertheless we know very little about his biography. We don’t know where he studied law, nor whether he ever was professor in the Studium founded by Frederic II in Neaples. Many members of his family were judges so as a judge certainly was him. All in all he was a practical jurist but with a noteworthy education. His name is famous for his comment on the Liber constitutionum Regni Siciliae (very soon considered the ‘glossa ordinaria’ to that law code). His introduction (Proemium) to the Gloss calls the most attention by scholars. Marinus defends the idea of a strong and centralised monarchy. He draws from Roman law the concept of an absolute power, concentrated in the hands of the prince. Such a prince, anyway, for Marinus is no more the emperor, but the king of a single country (Regnum): a king who is strong enough to not have to recognise any other ‘temporal’ authority above him. Marinus was interested non only in the sovereignty’s questions (non excluded the relationship with the Pope and the local autonomies, i.e. the commons), but also in fiscal system, in the feudal rules and in the exercise of the iurisdictio