Intact caveolae are required for proper extravillous trophoblast migration and differentiation

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium–nutrient mixture F12 and treated with 5 mM methyl-β-cyclodextrin (MβCD) to disrupt caveolae. We found that after MβCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MβCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.     

Genetic Characterization of a Fusarium Head Blight Resistance QTL from Triticum turgidum ssp. dicoccoides

Plant Molecular Biology Reporter - Qfhs.ndsu-3AS in wild emmer wheat (Triticum turgidum L. ssp. dicoccoides) is a major quantitative trait locus associated with type II resistance to Fusarium head...     

Adjustments of Protein Metabolism in Fasting Arctic Charr,Salvelinus alpinus

Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (K_S) in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems) were measured in the same fish. This study is the first to measure both K_S and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills). K_S was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins.     

Astrocytic Response in Hippocampus and Cerebral Cortex in an Experimental Epilepsy Model

Astrocytes are very sensitive to alterations in the brain environment and respond showing a phenomenon known as astroglial reaction. S100beta is an astroglial derived neurotrophic factor, seems to be involved in neuroplasticity. The aim of this work was to study the astrocytic response in rat hippocampus and cerebral cortex after repetitive seizures induced by 3-mercaptopropionic acid (MP) administration. Immunocytochemical studies were performed to analyze GFAP and S100beta expression. Both studied areas showed hypertrophied astrocytes with enlarged processes and increased soma size. Astrocyte hyperplasia was observed only in the cerebral cortex. A significant decrease in the astrocytic S100beta immunostaining occurs after MP treatment. These results indicate that MP administration induces an astroglial reaction with reduced intracellular S100beta level. The observed reduction in astroglial S100beta could be related to the release of this factor to the extracellular space, where it may produce neurotrophic or deleterious effects accordingly to the concentration achieved. The mechanism of this remains to be elucidated.     

Phosphatidylinositol 4,5-Bisphosphate Induced Flunitrazepam Sensitive-GABA<Subscript>A</Subscript> Receptor Increase in Synaptosomes from Chick Forebrain

The flunitrazepam sensitive-GABA_A receptor density was increased by cytochalasins C and D at 37°C suggesting that microfilament depolymerization induces exposure to the radioligand of a GABA_A receptor in synaptosomes (Pharm Biochem Behav 72 (2002) 497). Similarly, phosphatidylinositol-4,5-bisphosphate (1–5 μM), but not a mixture of phospholipids, induced an increase of GABA_A receptors in synaptosomes. Furthermore, N-ethyl maleimide, an inactivator of the sensitive fusion protein, which interacts with GABA_A receptor, abolished the receptor increase induced by phosphatidylinositol-4,5-bisphosphate. Together, the results suggest that phosphatidylinositol-4,5-bisphosphate, acts via microfilament depolymerization increasing the binding of the radioligand to receptors possibly by modulation of their interaction with proteins involved in trafficking and docking mechanisms.     

Paracellular permeability restricts airway epithelial responses to selectively allow activation by mediators at the basolateral surface

Respiratory pathogens and toxins often assault the lung from the airway lumen. Airway epithelia may initiate and amplify inflammation in response to these attacks, but under certain conditions confinement of inflammation to the airway lumen may be beneficial to the host. Accordingly, we hypothesized that airway epithelial polarity allows different responses to basolateral vs apical stimuli that may modulate inflammation. Using primary human airway epithelial cells differentiated at an air-liquid interface in culture, we found that responses to several cytokines required basolateral mediator application. In contrast, responses to Haemophilus influenzae occurred after either basolateral or apical interaction with airway epithelia. Experiments focused on IFN-gamma receptor polarity confirmed its predominant basolateral location in cultured airway epithelia as well as in normal human airway tissue. Furthermore, physical and pharmacologic disruption of barrier function in airway epithelia allowed responses to apical application of IFN-gamma and other cytokines. These in vitro studies directly correlated with experiments in mice in which an airway epithelial response to IFN-gamma injected into the airway lumen was seen only after disruption of barrier function. The results indicate that airway epithelia with intact barrier function restrict inflammatory responses by limitation of cell activation through requiring interaction of selected mediators with the basolateral surface. However, loss of barrier integrity allows epithelial responses to these mediators if located in the airway lumen to amplify airway defenses.     

Lifeguard/neuronal membrane protein 35 regulates Fas ligand-mediated apoptosis in neurons via microdomain recruitment

Fas ligand (FasL)-receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up-regulated during postnatal development and in the adult. We show that over-expression of LFG protected cortical neurons from FasL-induced apoptosis and decreased caspase-activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL-induced cell death and caspase-8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL-induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL-mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL-activated signaling pathways.     

Schistosoma bovis: plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument

Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the male-but not female-S. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein extract of the worm tegument (TG) had the capacity to generate plasmin and to enhance the plasmin generation by the tissue-type plasminogen activator. Proteomic analysis of the TG extract identified 10 plasminogen-binding proteins, among which the major ones were enolase, glyceraldehyde-3-phosphate dehydrogenase and actin. This study represents the first report about the binding of plasminogen to Schistosoma sp. proteins.     

Specificity of olfactory receptor interactions with other G protein-coupled receptors

Studies on olfactory receptor (OR) pharmacology have been hindered by the poor plasma membrane localization of most ORs in heterologous cells. We previously reported that association with the beta(2)-adrenergic receptor (beta(2)-AR) facilitates functional expression of the OR M71 at the plasma membrane of HEK-293 cells. In the present study, we examined the specificity of M71 interactions with other G protein-coupled receptors (GPCRs). M71 was co-expressed in HEK-293 cells with 42 distinct GPCRs, and the vast majority of these receptors had no significant effect on M71 surface expression. However, co-expression with three subtypes of purinergic receptor (P2Y(1)R, P2Y(2)R, and A(2A)R) resulted in markedly enhanced plasma membrane localization of M71. Agonist stimulation of M71 co-expressed with P2Y(1)R and P2Y(2)R activated the mitogen-activated protein kinase pathway via coupling of M71 to Galpha(o). We also examined the ability of beta(2)-AR, P2Y(1)R, P2Y(2)R, and A(2A)Rto interact with and regulate ORs beyond M71. We found that co-expression of beta(2)-AR or the purinergic receptors enhanced the surface expression for an M71 subfamily member but not for several other ORs from different subfamilies. In addition, through chimeric receptor studies, we determined that the second transmembrane domain of beta(2)-AR is necessary for beta(2)-AR facilitation of M71 plasma membrane localization. These studies shed light on the specificity of OR interactions with other GPCRs and the mechanisms governing olfactory receptor trafficking.