Cell volume-dependent phosphorylation of proteins of the cortical cytoskeleton and cell-cell contact sites. The role of Fyn and FER kinases

Cell volume affects diverse functions including cytoskeletal organization, but the underlying signaling pathways remained undefined. We have shown previously that shrinkage induces Fyn-dependent tyrosine phosphorylation of the cortical actin-binding protein, cortactin. Because FER kinase was implicated in the direct phosphorylation of cortactin, we investigated the osmotic responsiveness of FER and its relationship to Fyn and cortactin. Shrinkage increased FER activity and tyrosine phosphorylation. These effects were abolished by the Src family inhibitor PP2 and strongly mitigated in Fyn-deficient but not in Src-deficient cells. FER overexpression caused cortactin phosphorylation that was further enhanced by hypertonicity. Exchange of tyrosine residues 421, 466, and 482 for phenylalanine prevented cortactin phosphorylation by hypertonicity and strongly decreased it upon FER overexpression, suggesting that FER targets primarily the same osmo-sensitive tyrosines. Because constituents of the cell-cell contacts are substrates of Fyn and FER, we investigated the effect of shrinkage on the adherens junctions. Hypertonicity provoked Fyn-dependent tyrosine phosphorylation in beta-catenin, alpha-catenin, and p120(Cas) and caused the dissociation of beta-catenin from the contacts. This process was delayed in Fyn-deficient or PP2-treated cells. Thus, FER is a volume-sensitive kinase downstream from Fyn, and the Fyn/FER pathway may contribute to the cell size-dependent reorganization of the cytoskeleton and the cell-cell contacts.     

Evaluation of a 4-aminopiperidine replacement in several series of CCR5 antagonists

The bicyclic 5-amino-3-azabicyclo[3.3.0]octanes were shown to be effective replacements for the conformationally restricted 4-aminopiperidine ring found in several series of CCR5 antagonists.     

Synthesis of very long chain (up to 36 carbon) tetra, penta and hexaenoic fatty acids in retina.

The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the other (mainly VLCPUFA), incorporate most of the label found in lipids. Phosphatidylcholine (PC) is the most highly labelled lipid class, since both types of 14C-labelled fatty acids, but especially this novel series of VLCPUFA, are concentrated in this phospholipid. Radioactivity from [14C]acetate is found in very long chain tetra, penta and hexaenoic fatty acids of PC. The labelling of 20:4(n - 6), 20:5(n - 3), 22:5(n - 6) and 22:6(n - 3) is much lower than that of longer polyenes of each of these series, indicating that VLCPUFA are synthesized in situ by successive elongations of the above polyenes, pre-existing in retina lipids. In various subcellular fractions isolated from retinas after incubations with [14C]acetate (including cytosol, microsomes, mitochondria and photoreceptor membranes), the labelling of the VLCPUFA of PC is very high, even at relatively short intervals of incubation. The results suggest that not only the synthesis but also the intracellular traffic among membranes of VLCPUFA-containing species of PC are very active processes in the retina.     

Hematologic effects of cytauxzoonosis in Florida panthers and Texas cougars in Florida.

Cytauxzoon felis is a long-recognized hemoparasite of free-ranging Florida panthers (Puma concolor coryi), but its prevalence and effect on the population has not been assessed. Red blood cell indices and white blood cell counts were compared between infected and noninfected Florida panthers and Texas cougars (Puma concolor stanleyana) from 1983-1997 in Florida (USA). The prevalence of cytauxzoonosis for both populations was 39% (11/28) for Texas cougars, 35% for Florida panthers (22/63) and 36% overall. Thirteen hematologic parameters were compared between C. felis positive and negative panthers and cougars. Florida panthers had significantly lower mean cell hemoglobin count (MCHC) and higher white blood cell (WBC), neutrophil, monocyte and eosinophil counts (P < or = 0.05) than Texas cougars. Infected Florida panthers had significantly lower mean cell hemoglobin (MCH) and monocyte counts and higher neutrophil and eosinophil counts than infected Texas cougars. Although statistically significant differences were measured for hematologic parameters in C. felis positive panthers and cougars, biologically significant differences were not likely because values were generally within expected reference ranges for healthy animals. Cytauxzoonosis does not appear to have a negative effect on the hematologic parameters of chronically infected panthers and cougars. Potential transient changes during initial infection were not evaluated.     

Biogeography and taxonomy of extinct and endangered monk seals illuminated by ancient DNA and skull morphology

Extinctions and declines of large marine vertebrates have major ecological impacts and are of critical concern in marine environments. The Caribbean monk seal, Monachus tropicalis, last definitively reported in 1952, was one of the few marine mammal species to become extinct in historical times. Despite its importance for understanding the evolutionary biogeography of southern phocids, the relationships of M. tropicalis to the two living species of critically endangered monk seals have not been resolved. In this study we present the first molecular data for M. tropicalis, derived from museum skins. Phylogenetic analysis of cytochrome b sequences indicates that M. tropicalis was more closely related to the Hawaiian rather than the Mediterranean monk seal. Divergence time estimation implicates the formation of the Panamanian Isthmus in the speciation of Caribbean and Hawaiian monk seals. Molecular, morphological and temporal divergence between the Mediterranean and “New World monk seals” (Hawaiian and Caribbean) is profound, equivalent to or greater than between sister genera of phocids. As a result, we classify the Caribbean and Hawaiian monk seals together in a newly erected genus, Neomonachus. The two genera of extant monk seals (Monachus and Neomonachus) represent old evolutionary lineages each represented by a single critically endangered species, both warranting continuing and concerted conservation attention and investment if they are to avoid the fate of their Caribbean relative.     

Oxidative stress reprograms lipopolysaccharide signaling via Src kinase-dependent pathway in RAW 264.7 macrophage cell line

Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-kappa B translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-kappa B translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-kappa B translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-kappa B translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-kappa B translocation in oxidant-treated macrophages, this strategy had no effect on NF-kappa B translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion.     

Retinoid X receptor activation is essential for docosahexaenoic acid protection of retina photoreceptors

We have established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes survival of rat retina photoreceptors during early development in vitro and upon oxidative stress by activating the ERK/MAPK signaling pathway. Here we have investigated whether DHA turns on this pathway through activation of retinoid X receptors (RXRs) or by inducing tyrosine kinase (Trk) receptor activation. We also evaluated whether DHA release from phospholipids was required for its protective effect. Addition of RXR antagonists (HX531, PA452) to rat retinal neuronal cultures inhibited DHA protection during early development in vitro and upon oxidative stress induced with Paraquat or H₂O₂. In contrast, the Trk inhibitor K252a did not affect DHA prevention of photoreceptor apoptosis. These results imply that activation of RXRs was required for DHA protection whereas Trk receptors were not involved in this protection. Pretreatment with 4-bromoenol lactone, a phospholipase A₂ inhibitor, blocked DHA prevention of oxidative stress-induced apoptosis of photoreceptors. It is noteworthy that RXR agonists (HX630, PA024) also rescued photoreceptors from H₂O₂-induced apoptosis. These results provide the first evidence that activation of RXRs prevents photoreceptor apoptosis and suggest that DHA is first released from phospholipids and then activates RXRs to promote the survival of photoreceptors.     

The shaping of intrinsic membrane potential oscillations: positive/negative feedback,ionic resonance/amplification,nonlinearities and time scales

The generation of intrinsic subthreshold (membrane potential) oscillations (STOs) in neuronal models requires the interaction between two processes: a relatively fast positive feedback that favors changes in voltage and a slower negative feedback that opposes these changes. These are provided by the so-called resonant and amplifying gating variables associated to the participating ionic currents. We investigate both the biophysical and dynamic mechanisms of generation of STOs and how their attributes (frequency and amplitude) depend on the model parameters for biophysical (conductance-based) models having qualitatively different types of resonant currents (activating and inactivating) and an amplifying current. Combinations of the same types of ionic currents (same models) in different parameter regimes give rise to different types of nonlinearities in the voltage equation: quasi-linear, parabolic-like and cubic-like. On the other hand, combinations of different types of ionic currents (different models) may give rise to the same type of nonlinearities. We examine how the attributes of the resulting STOs depend on the combined effect of these resonant and amplifying ionic processes, operating at different effective time scales, and the various types of nonlinearities. We find that, while some STO properties and attribute dependencies on the model parameters are determined by the specific combinations of ionic currents (biophysical properties), and are different for models with different such combinations, others are determined by the type of nonlinearities and are common for models with different types of ionic currents. Our results highlight the richness of STO behavior in single cells as the result of the various ways in which resonant and amplifying currents interact and affect the generation and termination of STOs as control parameters change. We make predictions that can be tested experimentally and are expected to contribute to the understanding of how rhythmic activity in neuronal networks emerge from the interplay of the intrinsic properties of the participating neurons and the network connectivity.