Taurine and guanidinoethanesulfonic acid (GES) differentially affects the expression and phosphorylation of cellular proteins in C6 glial cells.

Effects of taurine and guanidinoethanesulfonic acid (GES), a taurine transport inhibitor, on the expression and phosphorylation of cellular proteins in C6 glial cells were examined using two-dimensional (2-D) gel electrophoresis and 2-D immunoblots. 2-D gels stained with Coomassie Blue or SYPRO Ruby showed differential distribution patterns of cellular proteins in the control, taurine-supplemented and GES-supplemented cells. 2-D immunoblot analysis using the anti-phosphotyrosine antibody recognized only few immuno-reactive proteins in all three samples, and their distribution patterns were different. On the other hand, 2-D immunoblot analysis using the anti-phosphothreonine antibody recognized many immuno-reactive proteins with distinctly different distribution patterns in the control, taurine-supplemented and GES-supplemented cells. In GES-supplemented cells, the relative number of the anti-phosphotyrosine immuno-reactive protein spots increased modestly, whereas the relative number of the anti-phosphothreonine immuno-reactive protein spots decreased markedly than those in the control and taurine-supplemented cells.

Inhibition of taurine transport by cyclosporin A is due to altered surface abundance of the taurine transporter and is reversible.

We have investigated the underlying mechanism of the CsA-induced inhibition of taurine transport using a cell line permanently expressing the mouse taurine transporter (mTauT) tagged with the green-fluorescence protein (GFP). CsA inhibited the uptake activity of the expressed mTauT.GFP fusion protein in both dose and time dependent manner. Surface biotinylation assay revealed that the CsA-treatment reduced the relative surface abundance of the taurine transporter without affecting its total expression level. CsA treatment reduced both the taurine uptake and the relative surface abundance of the transporter by similar magnitudes. Conversely, when the CsA was washed off, both the uptake and the relative surface abundance of the transporter recovered fully to the control level. Remarkably, the recovery process was insensitive to the protein synthesis inhibitor cycloheximide. These results suggested that the CsA inhibited taurine transport by altering the surface abundance, possibly by internalization of the expressed taurine transporters.

Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site.

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) with both the 2-N and the 13'-positions of dTC. Examination of a dTC analog with a negative charge at the 13'-position failed to reveal electrostatic interaction with charged side-chain substitutions at gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to gammaTyr(117). This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.

Cloning, expression, and biological activity of recombinant alpha-cinnamomin: toxicity to cranberry and other plant species.

Elicitins produced by the pathogenic fungi Phytophthora are known to exhibit The elicitin cinnamomin is of nonspecific toxicity to different solanaceous plant species. particular interest for its potential role in the hypersensitive-like cell death and in the biological response of cranberry plants to the fungal pathogen Phytophthora cinnamomi. In order to understand the biochemical steps of the Phytophthora root rot disease in cranberry, we investigated the alpha-cinnamomin-induced plant responses. Toxicity of alpha-cinnamomin, which shows a high degree of sequence homology to the alpha-elicitin group, was tested on Vaccinum macrocarpon, Nicotiana tabacum, Capsicum annuum, Lycopersicon esculentum, Lactuca sativa, and Phaseolus vulgaris plants. Gene corresponding to alpha-cinnamomin gene fused with maltose binding protein gene, was cloned into a pMALTEV expression vector, which was transformed into E. coli cells. Cells containing alpha-cinnamomin clones were cultured and extracted protein was purified on a maltose binding protein affinity column. Biological activity of alpha-cinnamomin fusion protein was examined on propagated plants and cuttings. In cranberry plants treated with cinnamomin necrotic hypersensitive-like response in the proximal areas of the leaf lamina of lower plant leaves was observed after 48-72 hr of incubation. Limited leaf necrosis observed days after application of low amounts of recombinant cinnamomin directly on the leaves of other plants indicates that the recombinant protein might be functioning as a toxin, capable of inducing aging accompanied by plant cell death.