A Novel RNA Synthesis Inhibitor, STK160830, Has Negligible DNA-Intercalating Activity for Triggering A p53 Response, and Can Inhibit p53-Dependent Apoptosis

RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses

A comprehensive validation of very early rule-out strategies for non-ST-segment elevation myocardial infarction in emergency departments:protocol for a multicentre prospective cohort study

Introduction: Recent advances in troponin sensitivity enabled early and accurate judgement of ruling-out myocardial infarction, especially non-ST elevation myocardial infarction (NSTEMI) in emergency departments (EDs) with development of various prediction-rules and high-sensitive-troponin-based strategies (hs-troponin). Reliance on clinical impression, however, is still common, and it remains unknown which of these strategies is superior. Therefore, our objective in this prospective cohort study is to comprehensively validate the diagnostic accuracy of clinical impression-based strategies, prediction-rules and hs-troponin-based strategies for ruling-out NSTEMIs. Methods and analysis: In total, 1500 consecutive adult patients with symptoms suggestive of acute coronary syndrome will be prospectively recruited from five EDs in two tertiary-level, two secondary-level community hospitals and one university hospital in Japan. The study has begun in July 2018, and recruitment period will be about 1 year. A board-certified emergency physician will complete standardised case report forms, and independently perform a clinical impression-based risk estimation of NSTEMI. Index strategies to be compared will include the clinical impression-based strategy; prediction rules and hs-troponin-based strategies for the following types of troponin (Roche Elecsys hs-troponin T; Abbott ARCHITECT hs-troponin I; Siemens ADVIA Centaur hs-troponin I; Siemens ADVIA Centaur sensitive-troponin I). The reference standard will be the composite of type 1 MI and cardiac death within 30 days after admission to the ED. Outcome measures will be negative predictive value, sensitivity and effectiveness, defined as the proportion of patients categorised as low risk for NSTEMI. We will also evaluate inter-rater reliability of the clinical impression-based risk estimation. Ethics and dissemination: The study is approved by the Ethics Committees of the Kyoto University Graduate School and Faculty of Medicine and of the five hospitals where we will recruit patients. We will disseminate the study results through conference presentations and peer-reviewed journals

Involvement of protein kinase C in the UTP-mediated potentiation of cyclic AMP accumulation in mouse J774 macrophages

1. We have investigated the effects of nucleotide analogues on cyclic AMP formation in mouse J774 macrophages and the mechanisms involved. 2. UTP, in the concentration range 0.1–100 μM, induced concentration-dependent potentiation of prostaglandin E(1) (PGE(1))-induced cyclic AMP formation, but had no effect on basal cyclic AMP formation. UDP showed an equal potency, while 2-methylthio ATP, α,β-methylene ATP and β,γ-methylene ATP gave either a slight increase or had no effect at concentrations up to 100 μM. ATP, although 100 fold less effective than UTP, also caused cyclic AMP potentiation, but had no effect on agonist-stimulated or basal cyclic AMP levels. 3. The cyclic AMP potentiation effect of UTP correlated with increased [Ca(2+)](i) and inositol phosphate (IP) formation over the same concentration range. 4. Ionomycin, which evokes an increase in [Ca(2+)](i) without affecting IP formation, did not cause an increase in cyclic AMP content, indicating that UTP-induced cyclic AMP regulation is not due to activation of Ca(2+)-sensitive adenylyl cyclase isoforms. 5. Although reduced, UTP potentiation was seen in cells incubated in a Ca(2+)-free and/or BAPTA-containing medium. Under these conditions, the UTP-increased IP accumulation was similarly reduced. 6. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) also increased PGE(1) stimulation of cyclic AMP levels, and the UTP-induced potentiation of cyclic AMP formation was inhibited by either staurosporine or Ro 31-8220. Pretreatment of cells with PMA for 4–24 h resulted in marked attenuation of UTP-stimulated cyclic AMP potentiation. 7. Pretreatment with pertussis toxin (24 h, 100 ng ml(−1)) did not significantly affect UTP-induced cyclic AMP potentiation and IP formation, although it increased the cyclic AMP response to PGE(1). 8. Analysis of J774 cells by Western blotting with antibodies specific for different protein kinase C (PKC) isoforms shows the presence of the βI, βII, δ, ε, ζ, μ, λ and ζ isoforms. Moreover, UTP significantly increased the level of PKC βI, βII, δ, ε, μ, λ and ζ immunoreactivity in the membrane fraction and decreased the cytosolic reactivity of PKC βII, δ, ε and ζ. 9. Immunoblot studies also indicate the presence of type II adenylyl cyclase. 10. These results indicate that PKC is required for the potentiation of adenylyl cyclase activity by macrophage pyrimidinoceptors, which exhibit a higher specificity for UTP and UDP than for ATP