TFIID, one of the multiple eukaryotic general transcription factors (GTFs), plays a key role in DNA-dependent RNA polymerase II (RNAP II)-mediated transcription initiation and regulation. The form and function of TFIID have been extensively studied by using in vitro approaches (see references 7, 22, and 57 for recent reviews). TFIID exists as a stable multisubunit complex in a variety of distinct eukaryotic systems (7,17,22,47,57,92), and in vitro transcription assays have shown that one subunit of TFIID, the TATA-binding protein (TBP), is sufficient for TATA element DNA recognition and subsequent incorporation of the other GTFs and RNAP II into the preinitiation complex (PIC). A TBP-assembled PIC can catalyze basal transcription in vitro (6, 67). However, these same in vitro assays also demonstrate that activation of transcription by sequence-specific DNA binding transactivator proteins can be observed only from a PIC formed by utilizing the multisubunit TBP-containing complex, TFIID, and not with TBP (32, 64). This observation suggested that the other subunits of TFIID are essential for its regulatory functions and ultimately led to the identification of the TBP-associated factors (TAFs), which in combination with TBP comprise the TFIID complex (17,47,64,92).At least 8 to 10 RNAP II-specific TAFs (or TAF II s) associate with TBP to form eukaryotic TFIID. These TAF II s exhibit molecular masses ranging from 250 to 15 kDa, depending on the organism analyzed (human, Drosophila melanogaster, and Saccharomyces cerevisiae) (17,47,61,66,92; see reference 7 for a recent review). A comparison of the amino acid sequences of TAF II s from these evolutionarily divergent organisms shows that there is a striking conservation of TAF II sequences (7). The results of various biochemical analyses have led to the hypothesis that TAF II s participate in a variety of protein-protein and protein-DNA interactions. These interactions range from the facilitation of the formation and/or stabilization of the PIC by TAF II binding to DNA (78,82,83) to TAF II s interacting with each other (7, 29, 30, 33, 34, 37, 41-46, 53, 71, 79, 80, 83, 86, 87, 89, 91) or with basal transcription factors (7,23,26,30,42,90) to the direct interaction of TAF II s with the activation domains of transcriptional regulatory molecules (9, 11, 12, 19-21, 23, 31, 37, 42, 51, 72, 75, 80, 81, 86). Clearly, TAF II s are involved in a large number of critical regulatory events in RNAP II transcription, and detailed analyses of TAF II molecules will shed light on the molecular mechanisms of RNAP II transcriptional regulation.The largest subunit of metazoan TFIID has been termed either hTAF II 250 or dTAF II 250, depending on whether it is a human or Drosophila protein. The human protein, hTAF II 250, contains an acidic N terminus, a central region including a high-mobility-group (HMG) homology box, and two bromodomain-like direct repeats, as well as a glycine-and serine-rich C terminus (29, 71). The Drosophila homolog, dTAF II 250, has Ͼ90% similarity and Ͼ50% iden...

Abstract. Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.

Lassa fever is an acute febrile disease of West Africa, where there are as many as 300,000 infections a year and an estimated 3000 deaths. As control of the rodent host is impracticable at present, the best immediate prospect is vaccination. We tested as potential vaccines in rhesus monkeys a closely related virus, Mopeia virus (two monkeys), and a recombinant vaccinia virus containing the Lassa virus glycoprotein gene, V-LSGPC (four monkeys). Two monkeys vaccinated with the New York Board of Health strain of vaccinia virus as controls died after challenge with Lassa virus. The two monkeys vaccinated with Mopeia virus developed antibodies measurable by radioimmunoprecipitation prior to challenge, and they survived challenge by Lassa virus with minimal physical or physiologic disturbances. However, both showed a transient, low-titer Lassa viremia. Two of the four animals vaccinated with V-LSGPC had antibodies to both Lassa glycoproteins, as determined by radioimmunoprecipitation. All four animals survived a challenge of Lassa virus but experienced a transient febrile illness and moderate physiologic changes following challenge. Virus was recoverable from each of these animals, but at low titer and only during a brief period, as observed for the Mopeia-protected animals. We conclude that V-LSGPC can protect rhesus monkeys against death from Lassa fever.

Since apoptosis appeared to be related to neurodegenerative processes, neuroprotection has been involved in investigation of therapeutic approaches focused upon pharmacological agents to prevent neuronal programmed cell death. In this regard, erythropoietin (Epo) seems to play a critical role. The present work was focused on the study of the Epo protective effect upon human neuroblastoma SH-SY5Y cells subjected to differentiation by staurosporine. Under this condition, profuse neurite outgrowth was accompanied by programmed cell death (35% of apoptotic cells by Hoechst assay, showing characteristic DNA ladder pattern). A previous treatment with recombinant human Epo (rHuEpo) increased the expression of the specific receptor for Epo while prevented apoptosis. Simultaneously, morphological changes in neurite elongation and interconnection induced by staurosporine were blocked by Epo. These Epo effects proved to be associated to the induction of Bcl-xL at the mRNA and protein levels (RT-PCR and Western blot after immunoprecipitation) and were mediated by activation of pathways inhibited by wortmannin. In conclusion, the fact that both events induced by staurosporine, cell apoptosis and differentiation, were prevented in SH-SY5Y cells previously exposed to rHuEpo suggests interrelated signaling pathways triggered by the Epo/EpoR interaction.

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There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.

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