PubMed · 14110806
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T HASE. 1964. A SIMPLE MICROELECTROPHORETIC METHOD USING STARCH-AGAR GEL.. https://pubmed.ncbi.nlm.nih.gov/14110806/
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Previous work has shown that the Simian Virus 40 T antigen (T antigen) cannot transform mouse embryo fibroblasts (MEFs) that do not express the type 1 insulin-like growth factor receptor (IGF-IR). We have now investigated the mechanism(s) by which the transforming activity of T antigen is affected by IGF-IR signaling. We demonstrate that transformation by T antigen of MEFs and several other cell lines requires an insulin receptor substrate-1 (IRS-1) phosphorylated on tyrosines. If IRS-1 is not expressed, or is serine phosphorylated or otherwise inactive, T antigen fails to transform cells in culture. For instance, while T antigen cannot transform 32D myeloid cells (that do not express IRS-1), its transforming activity is restored by the expression of a wild-type IRS-1, but not of an IRS-1 mutated at the PI3K binding sites. The importance of IRS-1 activation of PI3K in T-antigen transformation is supported by the finding that a constitutively activated p110 subunit of PI3K, a target of IRS-1, overcomes the inability of T antigen to transform MEFs with a serine phosphorylated IRS-1. Taken together, these results indicate that the IRS-1/PI3K signaling is one of the mechanisms regulating transformation by the SV40 T antigen. We propose that the requirement for a tyrosyl-phosphorylated IRS-1 provides a mechanism to explain the failure of T antigen to transform MEFs with deleted IGF-IR genes.
PURPOSE: To elucidate whether BACTEC Peds Plus F broth, usually used for culturing body fluids in paediatric departments, can be used for corneal cultures from cases with clinically suspected infectious keratitis, and to compare yields between this method and traditional methods (blood agar, chocolate agar, a fungal media, and swab transport media). METHODS: All cases with newly diagnosed, nonviral, clinically suspected infectious keratitis with no prior antibiotic therapy, were cultured both in the BACTEC Peds Plus F broth and the traditional method. McNemar's test was used for pairwise comparisons of the rates of positive growth between the two groups. RESULTS: In total, 30 eyes were included in this study. The growth rates for the traditional method and the BACTEC broth were similar (50.0 and 53.33%, respectively, P=1.0). The overall growth rate for the two methods combined was 73.33%, which is 45.29% higher than the reported yield in the literature (average of 50.47%). CONCLUSIONS: Our results show that BACTEC Peds Plus F broth can be used successfully in the work-up of clinically suspected infectious keratitis. The method has, apparently, several advantages over the 'Traditional method:' time-savings, as only one medium needs to be inoculated, transportation to the laboratory is simpler as there is no need for immediate incubation, and there is no need to keep and maintain a supply of fresh agar media. This method is especially suitable for office settings and remote clinics, but also can be used in hospital setting, as an adjunct, when available, to increase the growth yield.
The c-Myc oncoprotein plays a central role in human cancer via its ability to either activate or repress the transcription of essential downstream targets. For many of the repressed target genes, down-regulation by c-Myc relies on its ability to bind and inactivate the transcription factor Miz-1. Although Miz-1 inactivation is suspected to be essential for at least some of the biological activities of c-Myc, it has been difficult to demonstrate this requirement experimentally. Using a combination of short hairpin RNA-mediated knockdown and a previously characterized mutant of c-Myc that is defective for Miz-1 inactivation, we examined whether this inactivation is critical for three of the most central biological functions of c-Myc, cell cycle progression, transformation, and apoptosis. The results of this analysis demonstrated that in the in vitro assays utilized here, Miz-1 inactivation is dispensable for c-Myc-induced cell cycle progression and transformation. In marked contrast, the ability of c-Myc to induce apoptosis in primary diploid human fibroblasts in response to growth factor withdrawal is entirely dependent on its ability to inactivate Miz-1. These data have a significant impact on our understanding of the biochemical mechanisms dictating how c-Myc mediates opposing biological functions, such as transformation and apoptosis, and demonstrate the first requirement for Miz-1 inactivation in any of the biological functions of c-Myc.