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Thrombocytopenia reduces the number of metastases produced by a wide variety of murine tumors. Studies aimed at investigating interactions between tumors and platelets reveal that many tumors aggregated platelets in vitro andlor produced thrombocytopenia in vivo. I n some instances, tumor-cell-induced thrombocytopenia in vivo was accompanied by accumulation of platelets in the lung. Thrombocytopenia was most active against metastases produced by tumors with the capacity to aggregate platelets

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) As summarized in the preceding paper (26), genetic and biochemical evidence indicates that the early region of the simian virus 40 (SV40) genome is required for many of the biological activities of the virus. This region is needed for viral DNA replication (35), autoregulation of early genes (1, 14, 29, 38), cell transformation (2,3,15,16,18,23,37), and stimulation of cellular DNA and RNA syntheses (4,9,12,13,19,22,27,30,31,34,39). Although the early region codes for two proteins, the large T antigen (containing 708 amino acids) and the small t antigen (containing 174 amino acids), most of these functions have been attributed to the large T antigen. As is true of many other proteins which exhibit multiple functions (for a review, see reference 17), some of the functions of the large T antigen can be localized to specific domains. These domains can be determined in some detail by studying the functions of modified, cloned SV40 DNAs introduced into the nuclei of cells by the manual microinjection technique of Graessmann and Graessmann (10). By microinjecting fragments of SV40 DNA, Galanti et al. (9) have previously been able to distinguish those sequences in the T-antigen gene that are critical for the stimulation of cell DNA replication from those that are critical for the activation of silent rRNA genes. In the present communication we report further experiments in which this technique was applied to the cloned deletion mutants of SV40 described in the preceding paper (26) to refine the map positions of T-antigen coding regions involved in these two activities. MATERIALS AND METHODSGrowth and preparation of cells. tsl3 cells, a Glspecific temperature-sensitive mutant originally derived from BHK cells (20), were grown at 34°C on glass cover slips as previously described. Before microinjection, tsl3 cells were made quiescent by allowing them to grow for 7 days in Dulbecco minimal essential medium supplemented with 1% calf serum. After microinjection, cells were incubated at 340C for 24 h in the same medium supplemented with [3H]thymidine (0.7 ,Ci/ml). All media were warmed to the temperature of incubation before use.

Trypanosoma cruzi is the etiological agent of Chagas disease. The parasite has to overcome oxidative damage by ROS/RNS all along its life cycle to survive and to establish a chronic infection. We propose that T. cruzi is able to survive, among other mechanisms of detoxification, by repair of its damaged DNA through activation of the DNA base excision repair (BER) pathway. BER is highly conserved in eukaryotes with apurinic/apirimidinic endonucleases (APEs) playing a fundamental role. Previous results showed that T. cruzi exposed to hydrogen peroxide and peroxinitrite significantly decreases its viability when co-incubated with methoxyamine, an AP endonuclease inhibitor. In this work the localization, expression and functionality of two T. cruzi APEs (TcAP1, Homo sapiens APE1 orthologous and TcAP2, orthologous to Homo sapiens APE2 and to Schizosaccaromyces pombe Apn2p) were determined. These enzymes are present and active in the two replicative parasite forms (epimastigotes and amastigotes) as well as in the non-replicative, infective trypomastigotes. TcAP1 and TcAP2 are located in the nucleus of epimastigotes and their expression is constitutive. Epimastigote AP endonucleases as well as recombinant TcAP1 and TcAP2 are inhibited by methoxyamine. Overexpression of TcAP1 increases epimastigotes viability when they are exposed to acute ROS/RNS attack. This protective effect is more evident when parasites are submitted to persistent ROS/RNS exposition, mimicking nature conditions. Our results confirm that the BER pathway is involved in T. cruzi resistance to DNA oxidative damage and points to the participation of DNA AP endonucleases in parasite survival.