Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.
Adaptor Proteins, Signal Transducing ; Amino Acid Sequence ; Animals ; COS Cells/enzymology ; Cercopithecus aethiops ; Enzyme Activation ; Epidermal Growth Factor/*pharmacology ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate/metabolism ; Molecular Sequence Data ; Phospholipase C/chemistry/*metabolism ; Phosphoric Monoester Hydrolases/chemistry/*metabolism ; Protein Binding ; Signal Transduction ; src Homology Domains/*physiology

OBJECTIVES: To circumvent the limitations of the current golden standard method, colony-forming unit (CFU) assay, for viability of Bacille Calmette–Guérin (BCG) vaccines, we developed a new method to rapidly and accurately determine the potency of BCG vaccines. METHODS: Based on flow cytometry (FACS) and fluorescein diacetate (FDA) as the most appropriate fluorescent staining reagent, 17 lots of BCG vaccines for percutaneous administration and 5 lots of BCG vaccines for intradermal administration were analyzed in this study. The percentage of viable cells measured by flow cytometry along with the total number of organisms in BCG vaccines, as determined on a cell counter, was used to quantify the number of viable cells. RESULTS: Pearson correlation coefficients of FACS and CFU assays for percutaneous and intradermal BCG vaccines were 0.6962 and 0.7428, respectively, indicating a high correlation. The coefficient of variation value of the FACS assay was less than 7%, which was 11 times lower than that of the CFU assay. CONCLUSION: This study contributes to the evaluation of new potency test method for FACS-based determination of viable cells in BCG vaccines. Accordingly, quality control of BCG vaccines can be significantly improved.
Administration, Cutaneous ; BCG Vaccine ; Cell Count ; Flow Cytometry* ; Fluorescein ; Methods ; Mycobacterium bovis ; Quality Control ; Stem Cells ; Vaccine Potency ; Vaccines*

NHERF1/EBP50 (Na⁺/H⁺ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.
Cell Membrane ; Cell Movement ; Cytoplasm ; Cytosol ; Epithelial Cells ; Membranes ; Ovarian Neoplasms* ; Pseudopodia ; Tumor Microenvironment