Biomedical subjects

J Lipecka

Publications and source records attributed to J Lipecka.

5 recordsLinked to original sources

PubMed2001

Regulation of ClC-2 chloride channels in T84 cells by TGF-alpha.

The almost ubiquitously expressed ClC-2 chloride channel is activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein tyrosine phosphorylation and activation of these channels. This study examines the possibility that transforming growth factor-alpha (TGF-alpha) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-alpha (0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a superimposed reversible activation of the current was observed at 8.3 nM TGF-alpha. Both effects required activation of the intrinsic epidermal growth factor receptor (EGFR) tyrosine kinase activity, of phosphoinositide 3-kinase, and of protein kinase C. With microspectrofluorimetry of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-alpha was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-alpha-induced alkalinization activates ClC-2 current. This study indicates that ClC-2 channels are targets for EGFR signaling in epithelial cells.

Biological Transport↗

PubMed2000

Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors.

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.

Cell Line, Transformed↗

PubMed1999

Aspirin and some other nonsteroidal anti-inflammatory drugs inhibit cystic fibrosis transmembrane conductance regulator protein gene expression in T-84 cells.

Cystic fibrosis (CF) is caused by mutations in the CF gene, which encodes CF transmembrane conductance regulator protein (CFTR), a transmembrane protein that acts as a cAMP-regulated chloride channel The disease is characterized by inflammation but the relationship between inflammation, abnormal transepithelial ion transport, and the clinical manifestations of CF are uncertain. The present study was undertaken to determine whether three nonsteroidal anti-inflammatory drugs (NSAIDs) (aspirin, ibuprofen, and indomethacin) modulate CFTR gene expression in T-84 cells. Treatment with NSAIDs reduced CFTR transcripts, and decreased cAMP-stimulated anion fluxes, an index of CFTR function. However, the two phenomena occurred at different concentrations of both drugs. The results indicate that NSAIDs can regulate both CFTR gene expression and the function of CFTR-related chloride transport, and suggest that NSAIDs act via multiple transduction pathways.

1-Methyl-3-isobutylxanthine↗

PubMed1998

New sequence-specific human ribonuclease: purification and properties.

A new sequence-specific RNase was isolated from human colon carcinoma T84 cells. The enzyme was purified to electrophoretical homogeneity by pH precipitation, HiTrapSP and Superdex 200 FPLC. The molecular weight of the new enzyme, which we have named RNase T84, is 19 kDa. RNase T84 is an endonuclease which generates 5'-phosphate-terminated products. The new RNase selectively cleaved the phosphodiester bonds at AU or GU steps at the 3' side of A or G and the 5' side of U. 5'AU3' or 5'GU3' is the minimal sequence required for T84 RNase activity, but the rate of cleavage depends on the sequence and/or structure context. Synthetic ribohomopolymers such as poly(A), poly(G), poly(U) and poly(C) were very poorly hydrolysed by T84 enzyme. In contrast, poly(I) and heteroribopolymers poly(A,U) and poly(A,G,U) were good substrates for the new RNase. The activity towards poly(I) was stronger in two colon carcinoma cell lines than in three other epithelial cell lines. Our results show that RNase T84 is a new sequence-specific enzyme whose gene is abundantly expressed in human colon carcinoma cell lines.

Adenocarcinoma↗

PubMed1996

Properties of a Cl(-)-conductive pathway(s) in microsomes from rat kidney inner medulla. Involvement of cystic fibrosis transmembrane regulator protein.

The properties of a protein-kinase-A(PKA)-activated Cl(-)-conductive pathway(s) in alkaline phosphatase-enriched microsomes from the rat inner medulla (IMV) were investigated. Transcripts of cystic fibrosis transmembrane regulator (CFTR) were detected by reverse transcription/polymerase analysis of total RNA from the inner medulla, while immunoblot analysis using anti-CFTR antibodies detected a 170-kDa protein in the IMV. The PKA Cl(-)-conductive pathway(s) was studied by measuring the rate of valinomycin-induced microsomal swelling by light scattering. PKA increased the rate of valinomycin-induced swelling of vesicles consistent with the presence of Cl(-)-conductive pathway(s). The pharmacological properties and anion selectivity of the PKA-activated Cl(-)-conductive pathway(s) were similar to those of the CFTR Cl(-) channel. Our results show that a CFTR Cl(-) channel and possibly another cAMP-activated pathway(s) may participate in Cl(-) secretion in the rat inner medulla.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗