When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.

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1. The affinity alkylation reaction of the cholinergic, depolarising ligand, bromoacetylcholine with reduced acetylcholine receptor in the membrane fragments of Torpedo marmorata and in Triton-solubilised receptor from cat denervated muscle has been studied.2. Brief pretreatment with 100 pM bromoacetylcholine abolishes all [3H]a-neurotoxin binding in both cases.3. In the receptor from each of these sources, the number of sites of specific a-neurotoxin binding is exactly equal to the number of sites that can be specifically alkylated by br~mol [~H]acetylcholine, at saturation of either ligand.4. The concentration-dependence of specific br~mo[~H]acetylcholine binding is found to be biphasic. A first phase can be clearly discerned in which one-half of the total specific ligand-binding sites are alkylated readily, and a second phase in which the remainder react at higher reagent concentrations. The same discrimination of two equal sets of ligand sites can be obtained by preblockade using low concentrations of unlabelled bromoacetylcholine followed by reaction with [3H]a-neurotoxin or bromo[3H]acetylcholine.5. In both phases, a single subunit of M , about 43000 is the sole site of specific alkylation in both Torpedo and muscle. The reasons for the appearance of two equal but distinct populations in the ligand binding sites in the receptors are discussed.A number of studies on the nicotinic acetylcholine receptor have endeavoured to quantify the number of binding sites for nicotinic ligands per molecule in this oligomeric protein [l -31. Measurements of the essentially irreversible binding of the specific antagonists, a-neurotoxins, to purified preparations of acetylcholine receptor, together with determination of their molecular weights, allow the number of toxin binding sites per oligomer to be estimated (for a review see [4]). However, the complicated pharmacology of the acetylcholine receptor complex [4], uncertainty in the exact receptor molecular weight, and readily reversible binding of quaternary ammonium effectors [5], are factors that have hindered the determination of the number of binding sites for Abbreviations. (cHxN)*C, dicyclohexylcarbodiimide; iPrZP-F, diisopropylfluorophosphate; T/L, stoichiometric ratio where T and L represent the total number of moles of toxin or ligand, respectively, that bind specifically to a given preparation of acetylcholine receptor.agonists. In practice, therefore, results have been related to a-toxin binding using a stoichiometric ratio TIL, where T and L represent the total number of moles of toxin or ligand, respectively, that bind specifically to a given preparation. Ligand binding in Torpedo has been investigated using the radiolabelled affinity reagent maleimido-benzyltrimethylammonium iodide [6], or more recently, bromoacetylcholine [7,8]. These reagents can specifically alkylate the acetylcholine receptor if a disulphide bond adjacent to the ligand binding site(s) has first been reduced [6,9] and studies on the Torpedo acetylcholine receptor have indicated a stoichio...

When stimulated to secrete HCl the gastric oxyntic cell undergoes profound morphological change. The identifiable apical cell surface is greatly expanded in the stimulated oxyntic cell as compared with nonsecreting ones. To account for this change, one hypothesis proposes that the expanded surface is derived from the fusion of cytoplasmic tubulovesicular membranes with the existing limited apical membrane surface. An alternative hypothesis suggests that the tubulovesicular compartment is actually confluent with the apical surface at all times and that the morphological appearance follows the expansion of this supercollapsed compartment as HCl secretion commences. A variety of morphological evidence is reviewed here including transmission electron microscopy during various stages of secretion and inhibition, analysis of freeze-fracture replicas, penetration of macromolecular tracers, and membrane surface-staining characteristics. It is concluded that the weight of evidence favors a membrane fusion process. Moreover, recent comparative studies of membrane fractions from resting and secreting stomachs show different morphological and functional properties that are also consistent with a fusion hypothesis as a fundamental event in the membrane transformation of the oxyntic cell.