Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3-y subunit of the TCR. To investigate the role of CD3'y phosphorylation in PKC-mediated TCR downregulation, point mutated CD3,y cDNA was transfected into the CD3'y-negative T cell line JGN and CD3'y transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3-y was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3'y truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3'y was required for PKC-mediated TCR downregulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKCmediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucineor tyrosine-based motifs is proposed.

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

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In the present study, water-soluble nonenzymatic browning products (melanoidins) formed in roasted malt were separated, quantified, and investigated for their effects on detoxifying mechanisms in intestinal Caco-2 cells. The melanoidins were prepared from roasted malt by hot water extraction, and the water-soluble compounds were separated into different molecular weight (MW) fractions by gel filtration chromatography. By monitoring the effluent at 300 nm, seven molecular fractions I-VII were consecutively collected, revealing that approximately 2.3% of the water-soluble compounds had mean MWs between 10000 and 30000 Da. Thus, the bulk of water-soluble malt melanoidins consisted of MW > 30000 Da, among which approximately 58% showed mean MWs between 60000 Da and 100000 Da, whereas approximately 32% exhibited mean MWs of 200000 Da. Biotransformation enzyme activities of NADPH-cytochrome c-reductase (CCR) and glutathione-S-transferase (GST) were analyzed in Caco-2 Cells after 48 h of exposure to the different MW fractions. The low MW fraction of 10000 Da was most effective in activating the CCR and the GST activities (+122 and +33% vs control, respectively). The majority of the mid molecular weight compounds tested showed an activating effect on CCR activity and an inhibitory effect on GST activity. These effects were most pronounced for compounds of up to 70000 Da and >200000 Da but less distinct for fractions of an average molecular weight of 100000 Da.

It is generally assumed that TCR assembly occurs in the endoplasmic reticulum (ER), and ER retention/degradation signals have been identified in several of the TCR chains. These signals are probably responsible for retention of incompletely assembled TCR complexes and free TCR chains in the ER. This study focused on the intracellular localization and transport of partially assembled TCR complexes as determined by confocal microscopy analyses. We found that none of the TCR chains except for TCR´were allowed to exit the ER in T cell variants in which the hexameric CD3 + 4 Ti § g CD3ˇ4 complex was not formed. Interestingly, TCRẃ as exported from the ER independently of other TCR chains and was predominantly located in a compartment identified as the Golgi apparatus. Furthermore, in the TCR´-negative cell line MA5.8, the hexameric CD3 + 4 Ti § g CD3ˇ4 complex was allowed to exit the ER and was also predominantly located in the Golgi apparatus. However, neither hexameric TCR complexes nor TCR´chains were efficiently expressed at the cell surface without the other. The observations that TCR´and hexameric TCR complexes are transported from the ER to the Golgi apparatus independently of each other and that these partial TCR complexes are unable to be efficiently expressed at the cell surface suggest that final TCR assembly occurs in the Golgi apparatus.