Anandamide (N-arachidonoyl-ethanolamine) was recently identified as a brain arachidonate derivative that binds to and activates cannabinoid receptors, yet the mechanisms underlying formation, release and inactivation of this putative messenger molecule are still unclear. Here we report that anandamide is produced in and released from cultured brain neurons in a calcium ion-dependent manner when the neurons are stimulated with membrane-depolarizing agents. Anandamide formation occurs through phosphodiesterase-mediated cleavage of a novel phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine. A similar mechanism also governs the formation of a family of anandamide congeners, whose possible roles in neuronal signalling remain unknown. Our results and those of others indicate therefore that multiple biochemical pathways may participate in anandamide formation in brain tissue. The life span of extracellular anandamide is limited by a rapid and selective process of cellular uptake, which is accompanied by hydrolytic degradation to ethanolamine and arachidonate. Our results thus strongly support the proposed role of anandamide as an endogenous neuronal messenger.

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The folding of a polypeptide chain of a relatively large globular protein into its unique three-dimensional and functionally active structure occurs via folding intermediates. These partly folded states of proteins are difficult to characterize, because they are usually short lived or exist as a distribution of possible conformers. A variety of experimental techniques and approaches have been utilized in recent years in numerous laboratories for characterizing folding intermediates that occur at equilibrium, including spectroscopic techniques, solution X-ray scattering, calorimetry and gel filtration chromatography, as well as genetic methods and theoretical calculations. In this review, we focus on the use of proteolytic enzymes as probes of the structure and dynamics of folding intermediates and we show that this simple biochemical technique can provide useful information, complementing that obtained by other commonly used techniques and approaches. The key result of the proteolysis experiments is that partly folded states (molten globules) of proteins can be sufficiently rigid to prevent extensive proteolysis and appear to maintain significant native-like structure.

Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.

Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, alpha-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to prompt its systematic use as a simple first step in the elucidation of structure-dynamics-function relationships of a novel and rare protein, especially if available in minute amounts.

High-throughput single-cell analysis is a challenging task. Label-free tomographic phase microscopy is an excellent candidate to perform this task. However, in-line tomography is very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions. We demonstrate that by exploiting the random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain in-line phase-contrast tomography, if smart strategies for wavefront analysis are adopted. In fact, surprisingly, a priori knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely retrieved through digital holography wavefront numerical analysis. This approach makes continuous-flow cytotomography suitable for practical operation in real-world, single-cell analysis and with a substantial simplification of the optical system; that is, no mechanical scanning or multi-direction probing is required. A demonstration is given for two completely different classes of biosamples: red blood cells and diatom algae. An accurate characterization of both types of cells is reported, despite their very different nature and material content, thus showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells.

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The protein toxin VacA, produced by cytotoxic strains of Helicobacter pylori, causes a vacuolar degeneration of cells, which eventually die. VacA is strongly activated by a short exposure to acidic solutions in the pH 1.5-5.5 range, followed by neutralization. Activated VacA has different CD and fluorescence spectra and a limited proteolysis fragmentation pattern from VacA kept at neutral pH. Moreover, activated VacA is resistant to pH 1.5 and to pepsin. The relevance of these findings to pathogenesis of H. pylori-induced gastrointestinal ulcers is discussed.