Inhibition of protein synthesis by the T cell receptor-inducible human TDAG51 gene product.

The T cell death associated gene 51 (TDAG51) was shown to be required for T cell receptor (TCR)-dependent induction of Fas/Apo1/CD95 expression in a murine T cell hybridoma. Despite the absence of a nuclear localization sequence and a nucleic acid binding domain, it was suggested to be localized in the nucleus and to function as a transcription factor regulating Fas-expression. However, we demonstrate that the human (h)TDAG51 protein is localized in the cytoplasm and the nucleoli, suggesting a role in ribosome biogenesis and/or translation regulation. Indeed, it strongly inhibited translation of a luciferase mRNA in a reticulocyte translational extract. Furthermore, cotransfection of hTDAG51 and the luciferase gene into 293T cells resulted in a strong inhibition of luciferase mRNA translation. Our findings were further strengthened by isolating in a yeast two-hybrid screen three proteins which are involved in the regulation of translation. We speculate that hTDAG51 couples TCR signaling to inhibition of protein biosynthesis in activated T lymphocytes.

Differentiating vector-derived mRNA from contaminating DNA templates by inverse RT-PCR.

Inverse RT-PCR was used for detecting doxycycline-induced mRNA expression after viral transduction with a retroviral vector harboring human TDAG51. After the circularization of double-stranded cDNA, induced transcripts originating from integrated vector genomes were selectively amplified, even in the presence of DNA templates. Thus, DNase treatment before amplification was unnecessary.

Ligation of cell surface CD4 inhibits activation-induced death of human T lymphocytes at the level of Fas ligand expression.

Cross-linking of cell surface CD4 molecules by anti-CD4 mAb or HIV-1 gp120/anti-gp120 Ab primes resting T lymphocytes for activation-induced cell death (AICD) triggered via the CD3/TCR complex. In striking contrast, we demonstrate here that preincubation of activated human CD4+ T cells with anti-CD4 mAb consistently inhibited AICD triggered via anti-CD3 mAb or Staphylococcus aureus enterotoxin A superantigen. Inhibition of AICD of CD4+ T cell clones was also observed with F(ab')2, but not with Fab, of anti-CD4 mAb. Moreover, soluble HIV-1 gp120, but not rIL-16, inhibited AICD stimulated by S. aureus enterotoxin A. In susceptible clones, CD4 ligation prevented the up-regulation of Fas ligand mRNA and cell surface expression in response to anti-CD3 mAb or superantigen stimulation. CD3/TCR-dependent protein tyrosine phosphorylation and cytokine production were also prevented by preceding CD4 ligation. The inhibition of AICD due to the prevention of Fas ligand upregulation reveals a novel immunoregulatory consequence of CD4 ligation that might play a role in HIV infection and in the therapeutic application of anti-CD4 mAb.

Reduced glutathione and S-acetylglutathione as selective apoptosis-inducing agents in cancer therapy.

The effect of reduced glutathione (GSH) and S-acetylglutathione (S-acglu) treatment on several tumor cell lines and normal cells in vitro was investigated. GSH and S-acglu applied at concentrations of 1 mM and 2 mM induced apoptosis in malignant cells as shown by DNA-fragmentation and staining of apoptotic cells with 7-amino-actinomycin D while viability and growth of normal cells were not significantly influenced by this treatment. The results demonstrated that GSH and S-acglu may be selective inducers of apoptosis in malignant cells.

The molecular basis for the natural resistance of the cytochrome bc1 complex from strobilurin-producing basidiomycetes to center Qp inhibitors.

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta-methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)-beta-methoxyacrylates.