DDR1 regulates the stabilization of cell surface E‐cadherin and E‐cadherin‐mediated cell aggregation

The stabilization of cell surface E‐cadherin is important for the maintenance of apical junction complexes and epithelial polarity. Previously, we reported that discoidin domain receptor 1 (DDR1) forms a complex with E‐cadherin at adhesive contacts; however, the regulatory role of DDR1 in the stabilization of cell surface E‐cadherin and E‐cadherin‐mediated cell behaviors remained undefined. To gain insight into these questions, we utilized two stable clones depleted for DDR1 via the small interfering RNA (siRNA) technique, and we over‐expressed DDR1 in MDCK cells. We performed Western blotting, immunofluorescence analysis, flow cytometry, and cell aggregation studies to investigate the effect of DDR1 on cell surface E‐cadherin. The results showed that both DDR1/2 and E‐cadherin use their extracellular domains to form DDR/E‐cadherin complexes. Neither the depletion nor the over‐expression of DDR1 changed the expression level of E‐cadherin in MDCK cells. Collagen disrupted the formation of E‐cadherin complexes and caused E‐cadherin to accumulate in the cytoplasm; however, over‐expression of DDR1 stabilized E‐cadherin on the cell surface and decreased its cytoplasmic accumulation. Furthermore, independently of collagen stimulation, the depletion of DDR1 resulted in a decrease in the level of cell surface E‐cadherin, which consequently caused its cytoplasmic accumulation and decreased E‐cadherin‐mediated cell aggregation. These results indicate that DDR1 can increase the stability of cell surface E‐cadherin and promote MDCK cell aggregation, which may be mediated through the formation of DDR1/E‐cadherin complexes. Overall, these findings have implications for the physiological roles of DDR1 in association with the maintenance of both the adhesion junction and epithelial polarity. J. Cell. Physiol. 224: 387–397, 2010. © 2010 Wiley‐Liss, Inc.     

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.     

Loss of escape-related giant neurons in a spiny lobster, Panulirus argus

When attacked, many decapod crustaceans perform tailflips, which are triggered by a neural circuit that includes lateral giant interneurons, medial giant interneurons, and fast flexor motor giant neurons (MoGs). Slipper lobsters (Scyllaridae) lack these giant neurons, and it has been hypothesized that behavioral (e.g., digging) and morphological (e.g., flattening and armor) specializations in this group caused the loss of escape-related giant neurons. To test this hypothesis, we examined a species of spiny lobster, Panulirus argus. Spiny lobsters belong to the sister taxon of the scyllarids, but they have a more crayfish-like morphology than scyllarids and were predicted to have escape-related giant neurons. Ventral nerve cords of P. argus were examined using paraffin-embedded sections and cobalt backfills. We found no escape-related giant neurons and no large axon profiles in the dorsal region of the nerve cord of P. argus. Cobalt backfills showed one fewer fast flexor motor neuron than in species with MoGs and none of the fast flexor motor neurons show any of the anatomical specializations of MoGs. This suggests that all palinuran species lack this giant escape circuit, and that the loss of rapid escape behavior preceded, and may have driven, alternative predator avoidance and anti-predator strategies in palinurans.     

MicroRNA-30a sensitizes tumor cells to cis-platinum via suppressing beclin 1-mediated autophagy

Autophagy is activated in cancer cells during chemotherapy and often contributes to tumor chemotherapy resistance. In this study, we characterized the role of microRNA-30a (miR-30a) in the coordination of cancer cell apoptosis and autophagy, which determines the sensitivity of cancer cells to chemotherapy. First, the autophagy activity in cancer cells increased after cis-dichloro-diamine platinum (cis-DDP) or Taxol treatment, as indicated by the enhanced expression of beclin 1, a key regulator of autophagy, and increased number of LC3-positive autophagosomes. Second, miRNA screening using a TaqMan probe-based quantitative RT-PCR assay identified that miR-30a, a miRNA that targets beclin 1, was significantly reduced in tumor cells by cis-DDP treatment. Forced expression of miR-30a significantly reduced beclin 1 and the autophagy activity of tumor cells induced by cis-DDP. Third, the blockade of tumor cell autophagy activity by miR-30a expression or 3-methyladenine significantly increased tumor cell apoptosis induced by cis-DDP treatment. Finally, an in vivo tumor implantation mouse model clearly showed that elevation of miR-30a in implanted tumor cells by administration of the recombinant lentivirus expressing miR-30a strongly enhanced cis-DDP-induced apoptosis of tumor cells. In conclusion, our results demonstrate for the first time that miR-30a can sensitize tumor cells to cis-DDP via reducing beclin 1-mediated autophagy and that increasing miR-30a level in tumor cells represents a novel approach to enhance the efficacy of chemotherapy during cancer treatment.     

Simultaneous detection of NADH and H₂O₂ using flow injection analysis based on a bifunctional poly(thionine)-modified electrode

We report here a novel detection scheme for simultaneous detection of NADH and H(2)O(2) based on a bifunctional poly(thionine)-modified electrode. Electropolymerization of thionine on a "preanodized" screen-printed carbon electrode effectively lowers the oxidation potential of NADH to 0.15 V (vs. Ag/AgCl). Since poly(thionine) is also a well known electrochemical mediator for H(2)O(2) reduction, we further developed a poly(thionine)-modified ring disk electrode for simultaneous measurement of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) by flow injection analysis. By applying the optimized detection potentials of 0.2V and -0.2V at disk and ring electrodes, respectively, this system allows the simultaneous measurement of both analytes with good sensitivity (0.13 μA/mM for H(2)O(2) and 0.34 μA/mM for NADH) and limit of detection (1.74 μM and 26.0 μM for NADH and H(2)O(2)). This opens the possibility of a whole series of biosensor applications.     

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion,Transendothelial Migration and Phagocytosis

Background

Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β₂ integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis.

Methodology/Principal Findings

THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β₂ integrin cell surface expression and β₂ integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β₂ integrins, in particular CD11b/CD18. SIRPα overexpression reduced β₂ integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β₂ integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression.

Conclusions/Significance

SIRPα negatively regulates β₂ integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.     

Functional succession of bacterioplankton on the basis of carbon source utilization ability by BIOLOG plates

Changes of bacterioplankton diversity in lake water were followed in triplicate, continuous-flow experimental tanks. Most probable numbers (MPN) were obtained for 95 different carbon sources using BIOLOG plates and were used to characterize bacterioplankton diversity. During 70 days of incubation, MPN declined for 15 of the 95 substrates while three of 95 appeared to be newly used, indicating functional succession in the bacterioplankton. Total bacterial cell abundance was constant from day 7 to day 70 of the incubation period. The succession of species composition of phyto- and zooplankton was also observed and suggested some involvement by phyto- and zooplankton species in the changes of bacterioplankton diversity. Thus, BIOLOG-based MPN assays is a simple but sensitive method for characterizing the changes in the bacterioplankton carbon utilization profile and is also useful for tracing the functional succession of bacterioplankton diversity within a community.