Di-lysine motif-like sequences formed by deleting the C-terminal domain of aquaporin-4 prevent its trafficking to the plasma membrane.

Aquaporin-4 is a transmembrane water channel protein, the C-terminal domain of which is facing the cytosol. In the process of investigating the role of the C-terminal domain of aquaporin-4 with regard to intracellular trafficking, we observed that a derivative of aquaporin-4, in which the C-terminal 53 amino acids had been removed (Δ271-323), was localized to intracellular compartments, including the endoplasmic reticulum, but was not expressed on the plasma membranes. This was determined by immunofluorescence staining and labeling of the cells with monoclonal antibody specifically recognizing the extracellular domain of aquaporin-4, followed by confocal microscopy and flow cytometry. Deletion of additional amino acids in the C-terminal domain of aquaporin-4 led to its redistribution to the plasma membrane. This suggests that the effect of the 53-amino acid deletion on the subcellular localization of aquaporin-4 could be attributed to the formation of a signal at the C terminus that retained aquaporin-4 in intracellular compartments, rather than the loss of a signal required for plasma membrane targeting. Substitution of the lysine at position 268 with alanine could rescue the Δ271-323-associated retention in the cytosol, suggesting that the C-terminal sequence of the mutant served as a signal similar to a di-lysine motif.

Evaluation of real-time PCR for quantitative detection of Escherichia coli in beach water.

The current investigation evaluated the use of real-time polymerase chain reaction (PCR) for quantitative detection of Escherichia coli in marine beach water. Densities of E. coli in 263 beach water samples collected from 13 bathing beaches in Hong Kong between November 2008 and December 2009 were determined using both real-time PCR and culture-based methods. Regression analysis showed that these two methods had a significant positive linear relationship with a correlation coefficient (r) of 0.64. Serial dilution of spiked samples indicated that the real-time PCR had a limit of quantification of 25 E. coli colonies in 100 mL water sample. This study showed that the rapid real-time PCR has potential to complement the traditional culture method of assessing fecal pollution in marine beach water.

Control of extravillous trophoblast function by the eotaxins CCL11, CCL24 and CCL26.

STUDY QUESTION: What are the effects of the eotaxin group of chemokines (CCL11, CCL24 and CCL26) on extravillous trophoblast (EVT) functions important during uterine decidual vessel remodelling? SUMMARY ANSWER: CCL11, CCL24 and CCL26 can regulate EVT migration, invasion and adhesion, highlighting a potential regulatory role for these chemokines during uterine decidual spiral arteriole remodelling in the first trimester of human pregnancy. WHAT IS KNOWN ALREADY: A successful human pregnancy depends on adequate remodelling of the uterine decidual spiral arterioles, a process carried out by EVT which invade from the placenta. The invasion by EVT into the maternal uterine decidual vessels is regulated by the interaction of many factors including members of the chemokine subfamily of cytokines. STUDY DESIGN, SIZE, DURATION: This study used the HTR8/SVneo cell line as a model for invasive EVT. All experiments were repeated on at least three separate occasions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effect of recombinant human CCL11, CCL24 and CCL26 on EVT migration and invasive potential was measured using the xCELLigence real-time system, wound-healing and Matrigel invasion assays, zymography to measure MMP activity and reverse zymography to measure TIMP activity. A commercially available adhesion assay was used to assess EVT adhesion to extracellular matrix proteins. MAIN RESULTS AND THE ROLE OF CHANCE: All the three eotaxins were found to significantly stimulate migration of the EVT-derived cell line HTR8/SVneo (P < 0.05) with no significant changes in cell number following treatment with each chemokine (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo invasion (P < 0.05) and MMP2 activity (P < 0.05) without any effects on TIMP2 activity (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo cell binding to collagen IV (P < 0.05) and fibronectin (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This work has been conducted in vitro with a commonly used cell line model of EVT, HTR8/SVneo. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first to comprehensively examine the effects of the eotaxin group of chemokines on EVT functions and demonstrates that all the three eotaxins have the ability to regulate EVT functions critical to their role in vessel remodelling. This identifies a new role for the eotaxin group of chemokines during placentation.