Correlation of cell cycle kinetics with p63 expression in human limbal epithelial cells expanded on intact human amniotic membrane.

Aim: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic.

Methods: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 microM 5-bromo-2'-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium.

Quantification of receptor-mediated endocytosis.

This method allows measuring of the receptor-mediated internalization of 125I-labeled conalbumin, the chicken egg white isoform of transferrin. Kinetic data, i.e.

Indirect immunofluorescence microscopy.

This is a standard protocol for the detection of intracellular proteins by indirect immunofluorescence microscopy in DT40. It has been used extensively to investigate the intracellular distribution of various proteins of the endocytic machinery.

Luciferase reporter assay.

This luciferase reporter assay provides a simple and highly sensitive method to determine the responsiveness of the Tet-system. It is quantitative, reproducible and easy to use in DT40 screens with both transient and stable transfections. The reaction catalyzed by firefly luciferase is the oxidation of luciferin in the presence of coenzyme A with concomitant production of a photon that can be measured by a luminometer as relative light units (RLU) or with a less sensitive scintillation counter.

Using DT40 to study clathrin function.

Clathrin-coated pits and vesicles play a major role in eukaryotic membrane trafficking pathways. We have used the DT40 system to delete endogenous clathrin genes selectively from DT40 and replace them with clathrin under the control of a tetracycline-regulatable promoter. This enabled clathrin expression to be manipulated, and the functional consequences of clathrin depletion to be studied in a stable vertebrate context.

Salbutamol inhibits trypsin-mediated production of CXCL8 by keratinocytes.

Treatment of primary keratinocytes (HEKAp) with trypsin led to the production and release of CXCL8. Production of CXCL8 was exquisitely sensitive to inhibition by co-treatment with the beta(2) agonist sabutamol (IC(50)=1.1 nM).

A dominant role for chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) in mediating chemotaxis of CRTH2+ CD4+ Th2 lymphocytes in response to mast cell supernatants.

Human cultured mast cells, immunologically activated with immunoglobuin E (IgE)/anti-IgE, released a factor(s) that promoted chemotaxis of human CRTH2+ CD4+ T helper type 2 (Th2) lymphocytes. Mast cell supernatants collected at 20 min, 1 hr, 2 hr and 4 hr after activation caused a concentration-dependent increase in the migration of Th2 cells. The effect of submaximal dilutions of mast-cell-conditioned media was inhibited in a dose-dependent manner by ramatroban (IC50 = 96 nm), a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), but not by the selective TP antagonist SQ29548, implicating CRTH2 in mediating the chemotactic response of these Th2 cells.

Prostaglandin D2 causes preferential induction of proinflammatory Th2 cytokine production through an action on chemoattractant receptor-like molecule expressed on Th2 cells.

PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells.

Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase.

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo-AP2 interactions occur via the mu2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for mu2 phosphorylation is in cargo recruitment because mu2 phosphorylation enhances its binding to internalization motifs.

Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes.

We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase-mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation.