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.     

ACE and LRPAP1 insertion-deletion polymorphisms in a northern Ivory Coast population

The ACE and the LRPAP1 gene insertion-deletion polymorphisms were determined in 133 healthy individuals sampled from Ouangolodougou, a village located in northern Ivory Coast. No sex differences were found in ACE and LRPAP1 gene frequencies. The ACE insertion and deletion alleles had frequencies of 0.346 and 0.654, respectively. The ACE gene was not in Hardy-Weinberg equilibrium because of an excess of heterozygote genotypes and a deficiency of I/I genotypes compared to the expected values. Statistical analysis showed a significantly lower frequency of I/I genotypes in the Ivory Coast population compared to Sudan, Kenya, African Americans, and African Brazilians (p < 0.05), whereas no differences were found with respect to Somalia. Conversely, the frequencies of the insertion and deletion alleles in the Ivorian population did not differ from those of other African populations. The LRPAP1 insertion and deletion allele frequencies found in our study (0.192 and 0.808, respectively) did not differ significantly from the Czech and Spanish populations, the only two populations previously characterized for this polymorphism. However, the frequency of the I/I genotype was significantly lower than the frequencies observed in the European samples. Because of the limited information on the LRPAP1 gene polymorphism distribution in worldwide populations, it was not possible to draw any conclusion.     

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.     

Biogenic amine actions on cholangiocyte function

Biogenic amines, such as serotonin, histamine, dopamine, and the catecholamines epinephrine and norepinephrine, regulate a multitude of cellular responses. A great deal of effort has been invested into understanding the effects of these molecules and their corresponding receptor systems on cholangiocyte secretion, apoptosis, and growth. This review summarizes the results of these efforts and highlights the importance of these regulatory molecules on the physiology and pathophysiology of cholangiocytes.