We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

During reproduction the mass and number of cells in the uterus and the mammary gland increase rapidly and then diminish more rapidly after their reproductive functions are completed. The diminishment of tissue mass, known as involution, involves an ordered series of events that includes apoptosis of resident cells, neutrophil invasion, the release of degradative enzymes and phagocytosis of cellular debris. Local signals are believed to regulate the progression of involution in each tissue. Here we show that the mammary gland and uterus express high levels of uterocalin, a protein that specifically induces apoptosis in neutrophils and other leucocytes. In the mammary gland, uterocalin expression is induced by weaning. In both tissues, uterocalin is expressed at extremely high levels such that it constitutes an average of 0.2-0.5% of the total extractable protein at its peak. Epithelial cells in the uterus and mammary gland produce uterocalin. In each case, the protein is secreted into the tissue lumen, with mammary-derived uterocalin being found in the milk. The period of highest uterocalin expression in vivo is consistent with the hypothesis that one of its physiological roles is to induce apoptosis of infiltrating neutrophils and thereby delay the entry of neutrophils into the tissue. It is proposed that the role of uterocalin during involution is to provide a window of time during which resident cells are protected from the degradative enzymes, free radicals and other secreted products of activated phagocytes to allow these cells to prepare to survive the processes of involution.

After their roles in reproduction are completed, the mass of the uterus and the mammary gland decrease rapidly by the process of involution that involves an ordered series of events including apoptosis, neutrophil entry, the release of degradative enzymes, and phagocytosis of cellular debris. The acute phase proteins are produced by the liver and other tissues in response to inflammation or a toxic challenge. Uterocalin (SIP24/24p3) is one of these proteins. During involution, the mammary gland and uterus express high levels of uterocalin that reach an average of 0.2-0.5% of the total extractable protein at its peak. Uterocalin and its orthologues have been demonstrated in vitro to (1). bind certain fatty acids and (2). specifically induce apoptosis in neutrophils and other leukocytes. The period of uterocalin expression during involution is consistent with the hypothesis that one of its physiological roles is to induce apoptosis of invading neutrophils and delay the entry of neutrophils into the tissue until the second phase of involution. Interestingly, it has been shown that uterocalin expression remains higher in primiparous gland than in virgin glands after the pregnant glands have completely involuted. This observation and the known protective effect of early pregnancy on later development of breast cancer suggest that the ability of uterocalin to induce apoptosis in neutrophils might also decrease oxidative and carcinogenic activity in the gland and result in a lower mutation rate and thus a lower probability of cancer in the primiparous gland.

While lipopolysaccharides (LPS) induce dendritic cell (DC) maturation and migration to lymph nodes, glucocorticoids such as dexamethazone (Dex) have a profound suppressive effect on immune response. The mechanisms that might control this suppressive effect of Dex have been extensively investigated in lymphocytes as possible targets. Much less is known on the effects of Dex on DC, although they are recognized to regulate immunity. To get insights into possible combined effects of Dex and LPS on DC functions, we have undertaken a genome-wide analysis of differentially expressed genes of DC treated with Dex alone, LPS alone, or both, using high-density oligonucleotide microarrays. Hierarchical clustering and principal component analysis (PCA) agreed in identifying 24 h as the time point that best discriminated the three treatments. Among the counteracting effects we have observed an inhibition of Dex on the LPS-induced upregulation of the chemokine receptor CCR7. In vivo, Dex treatment blocked the LPSinduced migration of DC, which lost their ability to reach the draining lymph nodes. In addition, we observed a synergistic effect of Dex and LPS on the expression of the secreted lipocalin 24p3, which has been reported to induce apoptosis in T cells and thus may be related to immune suppression.

The purpose of this study was to investigate the ability of astrocyte-derived factors to influence neural progenitor cell differentiation. We previously demonstrated that rat adult hippocampal progenitor cells (AHPCs) immunoreactive for the neuronal marker, class III β-tubulin (TUJ1) were significantly increased in the presence of astrocyte-derived soluble factors under non-contact co-culture conditions. Using whole cell patch clamp analysis, we observed that the co-cultured AHPCs displayed two prominent voltage-gated conductances - tetraethyl ammonium (TEA)-sensitive outward currents and fast transient inward currents. The outward and inward current densities of the co-cultured AHPCs were approximately 2.5-fold and 1.7-fold greater, respectively, than those of cells cultured alone. These results suggest that astrocyte-derived soluble factors induce neuronal commitment of AHPCs. To further investigate the activity of a candidate neurogenic factor on AHPC differentiation, we cultured AHPCs in the presence or absence of purified rat recombinant interleukin-6 (IL-6). We also confirmed that the astrocytes used in this study produced IL-6 by ELISA and RT-qPCR. When AHPCs were cultured with IL-6 for 6-7 days, the TUJ1-immunoreactive AHPCs and the average length of TUJ1-immunoreactive neurites were significantly increased, compared to the cells cultured without IL-6. Moreover, IL-6 increased the inward current density to a comparable extent as did co-culture with astrocytes, with no significant differences in the outward current density, apparent resting potential, or cell capacitance. These results suggest that astrocyte-derived IL-6 may facilitate AHPC neuronal differentiation. Our findings have important implications for understanding injury-induced neurogenesis and developing cell-based therapeutic strategies using neural progenitors.