Changes in the proteome of Salmonella enterica serovar Thompson as stress adaptation to sublethal concentrations of thymol

Thymol is a natural biocide and component of some essential oils from herbs. Its inhibitory effect on the growth of different microorganisms is well documented. The precise targets of the antibacterial action of thymol is not yet been fully established, the action seems to take place in different ways. The strain Salmonella enterica serovar Thompson MCV1 was grown in the presence of a sublethal concentration (0.01%) of thymol. The proteins extracted from treated and untreated cells were subjected to 2‐D PAGE, followed by in‐gel spot digestion and subsequent MALDI‐TOF analysis. The analysis of gels showed many proteins that were either upregulated or downregulated by the presence of thymol, with significant changes in proteins belonging to different functional classes. In particular, the thioredoxin‐1 was not expressed in the treated cells, indicating that its absence could be a consequence of the stress caused by the presence of thymol. On the other hand, different chaperon proteins were upregulated or de novo synthesis such as GroEL and DnaK, key proteins in the protection mechanism toward thermal stress. Outer membrane proteins were upregulated in treated cells; indeed the bacterial envelope stress response is trigged by the accumulation of misfolded outer membrane proteins. Moreover, the thymol seems to impair the citrate metabolic pathway, as well as many enzymes involved in the synthesis of ATP. Definitely, thymol plays a role in altering very different pathways of cell metabolism.     

Hydroxyapatite affinity chromatography for the highly selective enrichment of mono‐ and multi‐phosphorylated peptides in phosphoproteome analysis

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi‐phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono‐, di‐, tri‐, and multi‐phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.     

Dielectric Properties of the Plasma Membrane of Cultured Murine Fibroblasts Treated with a Nonterpenoid Extract of Azadirachta indica Seeds

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. In this report, we investigate the alterations of the biophysical properties of the plasma membrane caused by treatment with the nonterpenoid fraction of neem oil that we defined as methanolic extract (MEX). The dose-response effect was evaluated and a MEX-dependent cytoxicity evidenced. The effect of MEX on the plasma membrane was studied by a well-established dielectric spectroscopy technique: electrorotation, which allows single-cell analysis. Our results show a structural/functional alteration of the plasma membrane with an evident increase of specific capacitance and conductance. The biological implications of this effect are discussed.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Opposing actions of endocannabinoids on cholangiocarcinoma growth: recruitment of Fas and Fas ligand to lipid rafts

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G(i/o) protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G(i/o) protein-coupled receptor. Using the lipid raft disruptors methyl-beta-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.     

Degenerative and apoptotic events at retinal and optic nerve level after experimental induction of ocular hypertension

Ocular hypertension is a symptom of a glaucomatous condition characterized by a severe vision decrease. Blindness caused by the apoptotic death of the retinal ganglion cells and of the astrocytes of the optic nerve may eventually result. Experimental hypertension was induced by inoculation of methylcellulose in the anterior chamber. Chromatin staining, TUNEL assay, and inter-nucleosomal DNA fragmentation observed in retina and optic nerve strongly suggest that hypertension causes apoptosis. Immunolocalization of the fibrillary acidic glial protein, specific of cell stress, and caspase-3 in the same tissues, further support this mode of cell death. Activation of the ubiquitin dependant proteolytic system was also observed. Protection from apoptosis exerted by administration of the peroxide scavenger trolox, suggests that the apoptotic pathway is activated by an oxidative stress. The data presented here show that the experimental hypertensive insult induces degenerative and apoptotic events comparable to those observed in human glaucoma.     

Sleep apnea: epidemiology, pathophysiology, and relation to cardiovascular risk

Several studies have shown the occurrence of an independent association between obstructive sleep apnea syndrome (OSAS) and cardiovascular disease, including arterial hypertension, ischemic heart disease, and stroke. The pathogenesis of the cardiovascular complications of OSAS is still poorly understood, however. Several mechanisms are likely to be involved, including sympathetic overactivity, selective activation of inflammatory molecular pathways, endothelial dysfunction, abnormality in the process of coagulation, and metabolic dysregulation. The latter may involve insulin resistance and disorders of lipid metabolism. The aim of this review, which reports the data presented at a workshop jointly endorsed by the European Society of Hypertension and by the European Union COST action on OSAS (COST B26), is to critically summarize the evidence available to support an independent association between OSAS and cardiovascular disease.     

Telomere length status of somatic cell sheep clones and their offspring

This study was carried out to determine the telomere length status of sheep clones and their offspring, and to examine telomere dynamics and chromosomal abnormalities in culture propagated donor cells. Skin samples were collected from somatic cell nuclear transfer-derived sheep clones, and three of their progeny generated by natural mating. Samples were collected from control animals (n = 35), spanning in age from 1 month to 36 months of age. Genomic DNA was extracted from cell/tissue samples and their telomere lengths were assessed by terminal restriction fragment (TRF) analysis. Results revealed: that (a) sheep clones derived from cultured somatic cells have shortened telomere lengths compared to age-matched controls; (b) the offspring derived from natural mating between clones had normal telomere lengths compared to their age-matched counterparts; and donor cell cultures beyond 20 population doublings had significantly (P < 0.05) shortened telomeres and exhibited a higher numerical and structural chromosomal abnormalities.