Ion mobility-mass spectrometry reveals the influence of subunit packing and charge on the dissociation of multiprotein complexes

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.     

Technological use of donkey milk in cheesemaking

The efficacy of donkey milk as an alternative to hen's egg white lysozyme in preventing cheese blowing during ripening of Italian Grana cheeses was tested. Indices of proteolysis and lipolysis did not differ between the different cheesemaking processes. All tested products showed good microbiological and sensorial quality.     

Vitamin D3 Enriches Ceramide Content in Exosomes Released by Embryonic Hippocampal Cells

The release of exosomes can lead to cell–cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.     

Optimisation of the formulation and of the technological process of egg-based products for the prevention of Salmonella enteritidis survival and growth

The aim of this work was to study the survival of Salmonella enteritidis after a pressure treatment in relation to compositive variables (NaCl content, pH), both in model and real systems, the latter consisting in an egg-based mayonnaise type product. Moreover, the fate of the surviving cells of S. enteritidis has been monitored during storage at 10 degrees C and the growth or death parameters have been calculated and modelled in relation to pH, NaCl concentration of the medium and entity of the pressure treatment applied. The modelling of the effects of the environmental factors on the treatment effectiveness indicated that the salt content and pH displayed a synergistic effect with pressure, whose extent was higher in the mayonnaise based products than in BHI. In fact, while in the model systems the cell recovery and growth during the subsequent incubation at 10 degrees C was allowed in many combinations of the Central Composite Design, in the real systems no recovery or growth of S. enteritidis were observed. This viability loss, which was maximum at pH 4.00 or 2% NaCl, could not be attributed merely to the interactions of such variables, but probably involves the naturally occurring antimicrobial enzymes of the raw material, whose activity can be enhanced by the pressure treatment.     

Loss performance analysis of an ATM multiplexer loaded withhigh-speed on-off sources

The performance of an asynchronous transfer mode (ATM) multiplexer whose input consists of the superposition of a multiplicity of homogeneous on-off sources modeled by a two-state Markovian process is studied. The approach is based on the approximation of the actual input process by means of a suitably chosen two-state Markov modulated Poisson process (MMPP), as a simple and effective choice for the representation of superposition arrival streams. To evaluate the cell loss performance, a new matching procedure that leads to accurate results compared to simulation is developed. The application limits of the proposed method are also discussed. The outstanding physical meaning of this procedure permits a deep insight into the multiplexer performance behavior as the source parameters and the multiplexer buffer size are varied     

Ultrastructural study on human placenta from intrauterine growth retardation cases

A morphological study was performed on 27 human placentas from normal gestations (Group 1) and compared with those from eight cases of intrauterine growth restriction (IUGR) (Group 2). Semithin section light microscopy, transmission, and scanning electron microscopy were carried out on trophoblastic terminal villi, carefully identified under the stereomicroscope. In growth retardation cases, villi appear longer, thinner, and less vascularized, compared to the normal condition. Fibrinoid, an extracellular material of hematic origin, frequently fills villar stroma. The density of apical microvilli appears considerably reduced and occasional microvilli-free areas are observed in growth retardation cases. Moreover, the underlying basal membrane appears significantly thicker than that of normal syncytiotrophoblast. Recently, particular attention has been paid to apoptosis as a possible cell deletion mechanism in growth restriction. In our study, a majority of typical apoptotic features appear indifferently in both IUGR and normal pregnancy. Our data hints that growth retardation might be correlated with a complex of structural changes, suggestive of maternofetal traffic downregulation, but further studies are required to understand the underlying functional mechanisms.     

Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro

In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epithelium in vitro and may represent the signal which triggers release at ovulation in vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought about by capacitation. In this paper, the dynamics of intracellular free Ca2+concentration ([Ca2+]i) and protein tyrosine phosphorylation in sperm during and after heparin-induced release from in vitro cultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine were used to determine whether [Ca2+]i increase and/or hyperactivation can induce sperm release. Results showed that: 1. heparin-released sperm have significantly higher [Ca2+]i than adhering sperm; 2. heparin induces a [Ca2+]i elevation in the sperm head followed by detachment from the monolayers; 3. external Ca2+is not required for heparin-induced release; 4. [Ca2+]i increase and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably leading to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion.     

Variability within the Candida rugosa Upases family

Several fungi secrete lipase isozymes differing in biochemicalproperties and in some cases in substrate specificity. In theyeast Candida rugosa, a family of related genes encodes formultiple lipase proteins, highly homologous in sequence butpartially different in the regions interacting with the substratemolecule. Analysis of these substitutions performed on the basisof multiple alignments and using a 3-D model of the enzyme,allows identification of a restricted number of amino adds possiblyinvolved in substrate specificity of Candida lipases.     

Comprehension of cause€ffect relations in a tool-using task by chimpanzees (Pan troglodytes).

Five chimpanzees (Pan troglodytes) were tested to assess their understanding of causality in a tool task. The task consisted of a transparent tube with a trap-hole drilled in its middle. A reward was randomly placed on either side of the hole. Depending on which side the chimpanzee inserted the stick into, the candy was either pushed out of the tube or into the trap. In Experiment 1, the success rate of 2 chimpanzees rose highly above chance, but that of the other subjects did not. Results show that the 2 successful chimpanzees selected the correct side for insertion beforehand. Experiment 2 ruled out the possibility that their success was due to a distance-based associative rule, and the results favor an alternative hypothesis that relates success to an understanding of the causal relation between the tool-using action and its outcome. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Performance in a tool-using task by common chimpanzees (Pan troglodytes), bonobos (Pan paniscus), an orangutan (Pongo pygmaeus), and capuchin monkeys (Cebus apella).

Performance by individual animals of three species of great apes (Pan troglodytes, Pan paniscus, and Pongo pygmaeus) and capuchin monkeys (Cebus apella) was assessed by presenting a food treat inside a clear tube. The subjects readily used a straight stick to obtain the food. When sticks were bundled together, the apes immediately unwrapped the bundle to obtain an individual stick, whereas capuchins attempted to insert the bundled sticks. When a misshapen stick was provided, apes, but not capuchins, showed an improvement in terms of modifying the misshapen stick before insertion. Our results indicate that all these species can solve these tasks. However, only the performance of apes is consistent with emerging comprehension of the causal relations required for the avoidance of errors in the more complex tasks. (PsycINFO Database Record (c) 2010 APA, all rights reserved)