Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluorodUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human .8-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) catalyzes the conversion of 2'-deoxyuridine 5'-monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu, where H4PteGlu is tetrahydropteroylglutamic acid) to thymidine monophosphate (dTMP) and dihydrofolate (H2PteGlu) (1). This enzymatic reaction provides the sole intracellular de novo source of dTMP, and because of its central role in the synthesis of DNA precursors, TS remains an important target enzyme in cancer chemotherapy (2).Both the cDNA and corresponding mRNA clones for mouse (3) and human (4) TS have been isolated and sequenced, and these probes have facilitated the analysis of TS structure and expression and the study of the molecular basis of TS regulation. This enzyme has been purified and well characterized from various sources, including bacteria, bacteriophage, yeast, viruses, parasites, and mammals (5-9). TS is a dimeric protein with identical subunits, each =35 kDa, and comparison of the predicted primary amino acid sequence ofthe protein from eight different sources reveals that it is one of the most highly conserved proteins.Previous studies examining regulation of TS expression have concentrated on cell-cycle-directed events. Various investigators have shown that maximal TS activity occurs during periods of active DNA synthesis (10-12). Moreover, this increase in TS enzyme levels that arises as cells enter S phase appears to be regulated at both the transcriptional and posttranscriptional levels (13-15). Takeishi et al. (4) also suggested the possibility of translational regulation of TS expression given the theoretical potential of three interconvertible secondary structures, each containing a stem-loop structure in the 5' untranslated region (5' U...

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPIanchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPIanchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPIanchored proteins.

CNS involvement in PMLCL is associated with extranodal involvement other than bone marrow and may reflect the unique biology of this disease. The propensity to involve the CNS parenchyma raises the concern that intrathecal prophylaxis may not be effective and suggests that CNS imaging should be considered in patients with extranodal disease.

The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58±17 (mean±SD) and 5±2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182±34 and 26±6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing < 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 ;&M 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.