INH: We report two new approaches, using click-chemistry and disulfide bond bridges, to surface-immobilize nucleic acids for single-molecule fluorescence experiments using covalent bonds and self-assembled monolayers. Both approaches are specific and yield comparable results to the avidin-biotin linkage, but offer new surface chemical properties that might be advantageous to prevent non-specific binding of biopolymers to the surface and to expand the range of fluorescent probes that can be employed in single-molecule studies.
INH: We report two new approaches, using click-chemistry and disulfide bond bridges, to surfaceimmobilize nucleic acids for single-molecule fluorescence experiments using covalent bonds and self-assembled monolayers. Both approaches are specific and yield comparable results to the avidin-biotin linkage, but offer new surface chemical properties that might be advantageous to prevent non-specific binding of biopolymers to the surface and to expand the range of fluorescent probes that can be employed in single-molecule studies.
Intracellular Ca2þ signaling has a central role in regulation of salivary gland cell function. Coordination of Ca2þ signaling between cells contributes to synchronized and effective secretion of saliva. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca2þ waves (ICW) and their propagation in human salivary gland (HSG) cells were investigated using fura-2 fluorescence imaging. While not well understood, mechanical stimulation of a single cell in a cluster with a micropipette induces ICW. The Ca2þ signal is propagated from the stimulated cell to the 7-9th tier of cells or~120 mm. The following findings indicate that ICW propagation in HSG cells uses an extracellular and ATP-dependent pathway. The purinergic receptor antagonist suramin significantly decreased ICW propagation. Extracellular ATP or UTP abolished ICW suggestive of receptor desensitization. Gap junction intercellular communication is not involved in ICW in HSG cells because the gap junction inhibitor oleamide did not inhibit ICW. Furthermore, HSG cells showed poor dye coupling upon microinjection of Lucifer Yellow. The Ca2þ transients observed within each cell are dependent on Ca2þ release from the ER as thapsigargin abolished the ICW. The phospholipase C inhibitor U73122 also blocks ICW indicating that these transients are IP3-dependent. Furthermore, store operated Ca2þ entry (SOCE) modulates the amplitude of Ca2þ signal since removal of extracellular Ca2þ or a SOCE inhibitor SK&F 96365 decreased the amplitude of Ca2þ signal. Inhibition of mitochondrial Ca2þ uptake with FCCP/oligomycin or ruthenium red showed similar effects on the amplitude. These results indicate that propagation of this ICW utilizes extracellular ATP, likely through the P2U(P2Y2) receptor in HSG cells. The major Ca2þ mobilization mechanisms are IP3-dependent ER Ca2þ release and SOCE. Finally, mitochondrial energy metabolism and Ca2þ uptake modulated this ICW propagation.
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