ABSTRACT
Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.
Subject(s)
Adult Stem Cells/drug effects , Limbus Corneae/cytology , Receptor, Notch1/physiology , Receptor, Notch2/physiology , Adult , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Aged , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Differentiation/drug effects , Cells, Cultured , Diamines/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Middle Aged , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptor, Notch1/antagonists & inhibitors , Receptor, Notch1/biosynthesis , Receptor, Notch1/genetics , Receptor, Notch2/antagonists & inhibitors , Receptor, Notch2/biosynthesis , Receptor, Notch2/genetics , Thiazoles/pharmacology , Transcription Factor HES-1/biosynthesis , Transcription Factor HES-1/genetics , Young AdultABSTRACT
Lengthening smFRET lifetimes: We investigated various photoprotection system combinations to find the combination that optimally extended the photobleach lifetime of a Cy3/Cy5 smFRET pair attached to a DNA hairpin in a single-molecule environment. We found that the glucose/glucose oxygen-scavenging solution in combination with redox-based photostabilization solutions yielded the longest average photobleaching lifetimes.
Subject(s)
Carbocyanines/chemistry , Fluorescence Resonance Energy Transfer , Photobleaching , DNA/chemistry , Fluorescent Dyes/chemistry , Glucose/chemistry , Oxidation-ReductionABSTRACT
We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the "closed" cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.
