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The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.

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As shown previously by several authors, cycloheximide inhibits progesterone‐induced maturation. However, normal maturation can be obtained, in Xenopus, after a 5 h treatment with cycloheximide (10–20 μ/ml), followed by extensive washing. Under these conditions, protein synthesis is still 50% inhibited. When X.laevis oocytes are continuously treated with a progesterone and cycloheximide mixture, they undergo a degenerative process which has been termed ‘pseudomaturation’: the germinal vesicle membrane breaks down, but the chromosomes do not condense and the nucleoli do not disappear completely. Progesterone‐induced maturation was not arrested by fusidic acid in Rana pipiens, even after micro‐injection. Actinomycin D (10–20 μg/ml) speeds up maturation in both R. pipiens and X. laevis. Microinjection of α‐amanitin into control R. pipiens oocytes does induce maturation in a few cases. It is concluded that one of the effects of progesterone might be a repression of RNA synthesis. Successive treatments with cycloheximide and actinomycin D failed to induce maturation, but often produced ‘pseudomaturation’ in X. laevis. Injection of a cytoplasmic extract (centrifuged homogenate) from X. laevis eggs which have undergone maturation into recipient oocytes of the same species induces ‘pseudomaturation’ and strongly inhibits protein synthesis. If the membranes which surround the egg are eliminated before homogenization, true maturation is obtained. The membranous material apparently releases factors which exert a negative effect on maturation and protein synthesis. Homogenates from eggs which have not been treated with progesterone do not, after injection, induce ‘pseudomaturation’ and have little effect on protein synthesis. In contrast with the findings of Ecker and Smith (1971)11 on R. pipiens, protein synthesis in X. laevis, after a short stimulation, drops considerably (50%) during progesterone‐induced maturation.