Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of cleavage of complexes under the action of ultrasound. The specificity of ultrasound-induced cleavage of the DNA sugar-phosphate backbone was studied in actinomycin D (AMD) complexes with double-stranded DNA restriction fragments. After antibiotic binding, the cleavage intensity of phosphodiester bonds between bases was shown to decrease at the chromophore intercalation site and to increase in adjacent positions. The character of cleavage depended on the sequences flanking the binding site and the presence of other AMD molecules bound in the close vicinity. A comparison of ultrasonic and DNase I cleavage patterns of AMD-DNA complexes provided more detail on the local conformation and dynamics of the DNA double helix in both binding site and adjacent regions. The results pave the way for developing a novel approach to studies of the nucleotide sequence dependence of DNA conformational dynamics and new techniques to identify functional genome regions.

Indomethacin is a non-steroidal anti-inflammatory drug with clearly known side effects on the gastrointestinal tract. The purpose of the present study was to investigate whether eupatilin inhibit cell injury induced by indomethacin in cultured feline esophageal epithelial cells (EECs). EECs were used to investigate the ability of eupatilin to induce the expression of heat shock proteins (HSP27 and HSP70) and analyze its cytoprotective effect against indomethacin-induced damage. The treatment of EECs with indomethacin for 8 h decreased cell viability. Western blot analysis showed that the levels of HSPs gradually decreased in cells treated with indomethacin, while eupatilin treatment increased the levels of HSPs. When treated with both indomethacin and eupatilin, the levels of HSPs increased rapidly, and were maintained at 130-140%. In addition, treatment with the specific inhibitors of PTK, PKC, PLC, p38 MAPK, JNKs, and PI3K attenuated the eupatilin-induced expression of HSPs. Pretreatment of EECs with the inhibitors of protein synthesis, actinomycin D or cycloheximide, attenuated the cytoprotective effect of eupatilin on indomethacin-induced cell damage. Reactive oxygen species production was upregulated by indomethacin, but downregulated by eupatilin. Taken together, it was suggested that HSPs were partly responsible for the eupatilin-mediated cytoprotective activity against the indomethacin-induced damage in EECs.

The p53 protein is an inducer of apoptosis, acting as a transcriptional regulator of apoptotic genes. In a previous study, we found that actinomycin D and nutlin-3a (A + N) synergistically activate p53. To better understand the molecular consequences of this synergism, we incubated arrays of antibodies against apoptotic proteins with extracts of A549 cells in which p53 had been activated. We found that strong activation of p53, marked by serine 46 and 392 phosphorylation, was associated with inactivating phosphorylation of proapoptotic BAD protein on serine 136. Investigation of the source of this phosphorylation revealed that activation of p53 was associated with accumulation of PIM2, a survival kinase. The accumulation of PIM2 following treatment with A + N was suppressed in p53-knockdown cells. Others discovered that PIM2 was activated by cooperatively acting p53 molecules. Our results are consistent with this finding. Moreover, we found that in A549 cells, the treatment with A + N stimulated in p53-dependent fashion the expression of other high cooperativity p53 target genes, DRAXIN and H19. Activation of antiapoptotic H19 can mechanistically explain relatively low rate of apoptosis of A549 cells exposed to A + N. We conclude that PIM2, DRAXIN and H19 are efficiently stimulated by strongly activated p53 molecules, probably acting cooperatively.

Purpose Intermediate-risk rhabdomyosarcoma (RMS) includes patients with either nonmetastatic, unresected embryonal RMS (ERMS) with an unfavorable primary site or nonmetastatic alveolar RMS (ARMS). The primary aim of this study was to improve the outcome of patients with intermediate-risk RMS by substituting vincristine and irinotecan (VI) for half of vincristine, dactinomycin, and cyclophosphamide (VAC) courses. All patients received a lower dose of cyclophosphamide and earlier radiation therapy than in previous trials. Patients and Methods Patients were randomly assigned at study entry to either VAC (cumulative cyclophosphamide dose, 16.8 g/m2) or VAC/VI (cumulative cyclophosphamide dose, 8.4 g/m2) for 42 weeks of therapy. Radiation therapy started at week 4, with individualized local control plans permitted for patients younger than 24 months. The primary study end point was event-free survival (EFS). The study design had an 80% power (5% one-sided α-level) to detect an improved long-term EFS from 65% (with VAC) to 76% (with VAC/VI). Results A total of 448 eligible patients were enrolled in the study. At a median follow-up of 4.8 years, the 4-year EFS was 63% with VAC and 59% with VAC/VI ( P = .51), and 4-year overall survival was 73% for VAC and 72% for VAC/VI ( P = .80). Within the ARMS and ERMS subgroups, no difference in outcome by treatment arm was found. Severe hematologic toxicity was less common with VAC/VI therapy. Conclusion The addition of VI to VAC did not improve EFS or OS for patients with intermediate-risk RMS. VAC/VI had less hematologic toxicity and a lower cumulative cyclophosphamide dose, making VAC/VI an alternative standard therapy for intermediate-risk RMS.

Interleukin (IL)-18 plays an important role in liver ischemia/reperfusion (I/R) injury. We have previously demonstrated that remifentanil protects against liver I/R injury by upregulating the hepatic expression of IL-18-binding protein (IL-18BP), a natural IL-18 inhibitor. The current study was performed to further clarify the effects of remifentanil on IL-18BP expression in the liver as well as investigate the underlying mechanisms. In Sprague-Dawley (SD) rats, we demonstrated that remifentanil significantly increased the expression of IL-18BP in normal rat liver tissue over a 24-h time period with maximal expression at 24 h after treatment. The upregulation of remifentanil on IL-18BP expression displayed similar trends in in vitro cellular studies, including mouse primary hepatocytes, normal human hepatocyte LO2, and mouse hepatoma cells Hep1-6. In LO2 cells, preexposure of the cells to remifentanil significantly inhibited IL-18-activated p65 NF-κB phosphorylation, and the inhibition was absent when the cells were transfected with IL-18BP siRNA, indicating the functional effects of IL-18BP induced by remifentanil. Pretreatment with actinomycin D abolished remifentanil-induced upregulation of IL-18BP mRNA, suggesting that the induction occurred at the transcriptional level. This was further supported by the luciferase reporter assay, which demonstrated that remifentanil treatment significantly increased transcription of the IL-18BP promoter. Both western blot analysis and ChIP assays showed that STAT1 and C/EBP β were activated by remifentanil. Furthermore, remifentanil failed to upregulate IL-18BP expression after silencing STAT1 or C/EBP β gene expression. These findings demonstrate that remifentanil could upregulate hepatic IL-18BP expression through transcriptional activation of the IL-18BP promoter, and STAT1 and C/EBP β are two key transcriptional factors involved in this process.

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signalling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription.

The aim of the present study was to establish the role of platelet‑activating factor acetyl hydrolase (PAF‑AH) in the resolution phase of gout using an established in vitro mononuclear cell model. The effects of signalling pathway inhibitors on PAF‑AH secretion, as well as the effects of the common treatments hydrocortisone and colchicine, an antibody against the anti‑inflammatory cytokine transforming growth factor β1 (TGFβ1), and the transcriptional inhibitor actinomycin D, were also investigated. The effect of recombinant PAF‑AH on cytokine secretion by these cells was also determined. Human peripheral blood‑derived monocytes were isolated and differentiated into macrophages. Monocytes and macrophages were stimulated with monosodium monohydrate urate (MSU) crystals or lipopolysaccharide in the presence or absence of AEG3482 [a c‑Jun N‑terminal kinase (JNK) inhibitor], MG132 (a proteasome inhibitor), hydrocortisone or colchicine. Cultures were then analysed for PAF‑AH secretion using ELISA. A 6‑fold upregulation of PAF‑AH secretion was observed following macrophage exposure to MSU crystals for 24 h (29.3±6 vs. 5.4±0.3 ng/ml unstimulated; P<0.05). Following 72 h, PAF‑AH levels decreased significantly (11.1±1.8; P<0.01). Secretion was further enhanced following pre‑treatment with the JNK protein kinase inhibitor AEG3482 prior to MSU crystal stimulation (P<0.05) and was abrogated when cells were preincubated with actinomycin D or the proteasome inhibitor MG132 (50, 100 and 200 µM). The addition of recombinant PAF‑AH (2.5‑10 ng/ml) to MSU crystal‑stimulated immature monocyte cultures significantly decreased pro‑inflammatory interleukin (IL)‑1β (unstimulated 687±124 vs. stimulated 113±30 pg/ml) and IL‑6 secretion (unstimulated 590±50 vs. stimulated 182±19 pg/ml). Treatment of MSU crystal‑stimulated macrophages with hydrocortisone (2 µM) also significantly decreased PAF‑AH release (P<0.05). Neutralising anti‑TGFβ1 addition decreased PAF‑AH dose‑dependently with the highest inhibition observed at 1 µg/ml (P<0.05). The results implicated that PAF‑AH may have an anti‑inflammatory role in the resolution phase of gout.

The ribosomal RNA genes (rDNA) of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200-300 tandem repeat rDNA units that encode 18S, 5.8S, 2S, and 28S ribosomal RNAs (rRNAs) necessary for ribosome biogenesis. In arthropods like Drosophila, about 60% of the rDNA genes have R1 and/or R2 retrotransposons inserted at specific sites within their 28S regions; these units likely fail to produce functional 28S rRNA. We showed earlier that R2 expression increases upon nucleolar stress caused by the loss of the ribosome assembly factor, Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 elements are transcribed by Pol I. A series of RT-PCRs established read-through transcription by Pol I from the 28S gene region into R1. Sequencing the RT-PCR products confirmed the 28S-R1 junction and the expression of R1 elements within nucleolar rDNA rather than R1 elements known to reside in centromeric heterochromatin. Using a genome-wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we show that Pol I activity on R1 elements is negligible under normal non-heat shock conditions but increases upon heat shock. We propose that prior to heat shock Pol I pauses within the 5' end of R1 where we find a consensus 'pause button', and that heat shock releases Pol I for read-through transcription farther into R1.