Inhibition of proliferation and apoptosis by the transcriptional repressor Mad1. Repression of Fas-induced caspase-8 activation.

Mad1 is a member of the Myc/Max/Mad network of transcriptional regulators that play a central role in the control of cellular behavior. Mad proteins are thought to antagonize Myc functions at least in part by repressing gene transcription. To systematically examine the function of Mad1 in growth control and during apoptosis, we have generated U2OS cell clones that express Mad1 under a tetracyline-regulatable promoter (UTA-Mad1). Mad1 was induced rapidly and efficiently, localized to the nucleus, and bound to DNA as a heterodimer with Max. The induction of Mad1 reduced cellular growth and, more profoundly, inhibited colony formation of UTA-Mad1 cells. Conditioned medium neutralized this inhibitory effect implying that Mad1 function is regulated by extracellular signals. In addition Mad1 interfered with Fas-, TRAIL-, and UV-induced apoptosis, which coincided with a reduced activation of caspase-8 during Fas-mediated apoptosis in response to Mad1 expression. Furthermore, microinjection of Mad1-expressing plasmids into fibroblasts inhibited apoptosis induced by the oncoproteins c-Myc and E1A. Thus, Mad1 not only interferes with cellular proliferation but also with apoptosis, which defines a novel aspect of Mad1 function.

Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization.

In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.

Novel ribonucleic acid species in Eimeria nieschulzi are associated with RNA-dependent RNA polymerase activity.

Sporulated oocysts of Eimeria nieschulzi, E. tenella and E. acervulina were screened for the presence of putative viral nucleic acids. An RNA-dependent RNA polymerase activity in E. nieschulzi correlated with the presence of unknown nucleic acid species that were absent in the two other species of Eimeria. The novel nucleic acid species also served as templates for RNA-polymerase activity in in vitro synthesis of full-length labelled transcripts. These nucleic acid species were shown to be RNAse-sensitive and were suspected to represent the genomic RNA of a putative virus.