Recent cDNA characterization has predicted the existence of a new member of the transforming growth factor family, transforming growth factor-beta 3 (TGF beta 3). However, nothing is known about the biological activities of the TGF beta 3 protein, since it has not been purified from any natural sources. We report here the recombinant expression in mammalian cells and the purification to apparent homogeneity of human TGF beta 3. The TGF beta 3 was evaluated in comparison with purified TGF beta 1 and TGF beta 2 in several assays for its effects on stimulation or inhibition of proliferation of mammalian cells. These analyses revealed that TGF beta 3 exerts activities similar to the two other TGF beta species, but that there are distinct differences in potencies between the different TGF beta forms depending on the cell type and assay used.

Entamoeba histolytica was found to grow normally without producing glutathione and the main enzymes of glutathione metabolism, indicating that glutathione is not essential for many eukaryotic processes. This parasitic amoeba is an unusual eukaryote whose special features may help define the crucial functions of glutathione in those eukaryotes that do use it. Since Entamoeba histolytica lacks mitochondria and the usual aerobic respiratory pathways, the finding that it grows without glutathione and other evidence support the hypothesis that a primary function of glutathione in eukaryotes involves protection against oxygen toxicity associated with mitochondria and suggest that eukaryotes may have acquired glutathione metabolism at the time that they acquired mitochondria.

With the expectation that trypanosomal glutathione (GSH) plays a major protective role against the endogenous oxidant stress that results form high intracellular levels of H2O2, we sought to deplete Trypanosoma brucei brucei of their GSH through inhibition of its biosynthesis. Administration of buthionine sulfoximine (BSO), a reversible inhibitor of gamma-glutamylcysteine synthetase, to parasitemic mice resulted in a progressive decrease in trypanosome GSH content, such that parasites isolated after 5 h or BSO treatment contained 50% of normal values. When BSO administration was continued for 18 h (intraperitoneal injection of 4 mmol/kg every 1.5 h), parasitemias temporarily cleared. When inhibitory plasma levels of BSO were maintained for about 27 h, two out of six infected mice were cured and the rest had significantly prolonged survival. These findings demonstrate the potential value of GSH depletion for the treatment of trypanosomiasis.

We have cloned and sequenced the 5' untranslated region of the transforming growth factor-,83 mRNA as well as the adjacent genomic sequence. S1 nuclease analysis identified a single transcription start site. We have thus determined that the 5' untranslated region is about 1.1 kb long and contains 11 open reading frames. In vitro translation of the TGF-133 precursor coding sequence was markedly inhibited by the presence of the 5' untranslated region. Similarly, when the 5' untranslated region of TGF-,13 was introduced upstream of the coding sequence of chloramphenicol acetyltransferase, in vitro translation was inhibited. Furthermore, upon transfection into 293 cells, chloramphenicol acetyltransferase expression was inhibited by the 5' untranslated region of TGF-,B3. The degree of translational inhibition was inversely proportional to the amount of transfected DNA. Mutation analysis implicated multiple segments of the 5' untranslated region as contributing to the inhibitory effect. Deletion of much of the 5'-most 640 nucleotides, including 8 of the 11 upstream ATGs, relieved much but not all of the inhibitory influence of the 5' untranslated region of TGF-43 mRNA. The two upstream open reading frames closest to the initiator codon for the TGF-,33 coding sequence also decreased translational efficiency, since mutation of either ATG resulted in increased translation. Transfection results with T47-D cells, a cell line which expresses TGF-133 mRNA, were similar to those obtained with the 293 cell line. Thus, TGF-j33 mRNA is a recent example of an expanding group of growth-related mRNAs in which the 5' untranslated region contains upstream open reading frames and other sequences which inhibit translation. Members of the transforming growth factor-,B (TGF-P)family are homodimeric proteins that act as potent regulators of growth and differentiation for a variety of cell types (22,34,40). cDNA cloning has resulted in the polypeptide sequence determination of five homologous TGF-1 species, of which TGF-1l, -,B2, and -13 are synthesized by mammalian cells. These three TGF-1 species have different expression patterns in vivo (30, 31), and their mRNA levels are differentially regulated in some settings (la, 3, 10). The mRNAs of these TGF-,B types each encode a large secreted precursor that includes the C-terminal 112-amino-acid TGF-,B mature monomer sequence. The human TGF-,3 precursor cDNA sequence included 260 nucleotides upstream of the translation initiation codon (7,42). Within this segment of the 5' untranslated region were two open reading frames, one of which extended into the coding sequence for the TGF-,B3 precursor in a different reading frame.According to the scanning model for the initiation of translation in eukaryotic cells, the 40S ribosomal subunit and translation initiation factors bind to the 5' end of the mRNA, traverse the mRNA in the 3' direction, and at an AUG triplet in the appropriate nucleotide context, usually the first AUG, assemble the complete complex and begin protein synthesis (18)

Abstract. Transforming growth factor-/3 (TGF-~) is a potent mediator of cell proliferation and extracellular matrix formation, depending on the cell type and the physiological conditions. TGF-~ is usually secreted in a "latent" complex that needs activation before it can exert its effects. Several observations correlate increased expression of TGF-/31 with tumorigenesis. To evaluate the physiological relevance of increased TGF-/31 synthesis in tumor cells we established cell clones overexpressing TGF-/~I and observed the resulting physiological changes in TGF-~ overproducing cells in vitro and in vivo. As a model system we used the human E1A-transformed 293 tumor cells, which are insensitive to the direct growth modulatory effects of TGF-/L The selection of this cell line allows an assessment of physiological alterations independent of TGF-/~ induced proliferative changes.