Small-Molecule Targeting of RNA Polymerase I Activates a Conserved Transcription Elongation Checkpoint

Summary Inhibition of RNA polymerase I (Pol I) is a promising strategy for modern cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme, but how this exceptional response is enforced is not known. Here, we define key elements requisite for the response. We show that Pol I preinitiation factors and polymerase subunits (e.g., RPA135) are required for BMH-21-mediated degradation of RPA194. We further find that Pol I inhibition and induced degradation by BMH-21 are conserved in yeast. Genetic analyses demonstrate that mutations that induce transcription elongation defects in Pol I result in hypersensitivity to BMH-21. Using a fully reconstituted Pol I transcription assay, we show that BMH-21 directly impairs transcription elongation by Pol I, resulting in long-lived polymerase pausing. These studies define a conserved regulatory checkpoint that monitors Pol I transcription and is activated by therapeutic intervention.Peer reviewe

PRENATAL EXPOSURE TO MODAFINIL ALTERS LOCOMOTOR BEHAVIOUR AND LEUCOCYTE PHAGOCYTOSIS IN MICE

Background: Modafinil is a psychostimulant drug prescribed mainly for treatment of narcolepsy but is used as a “smart drug” by wide populations to increase wakefulness, concentration and overall mental performance. The aim of this study was to assess potential developmental toxicity of modafinil. Materials and methods: Pregnant female mice were given either saline or modafinil (50 mg/kg orally) from gestational day (GD) 3 to GD 10 and then a challenge dose on the GD 17. The male offspring were treated analogously at the age of 10 weeks. Changes in the spontaneous locomotor/exploratory behaviour and anxiogenic profile in the open-field test were assessed in naïve animals, after an acute and 8th modafinil dose and the challenge dose following a 7-day wash-out period. One month after completion of the behavioural study, the leukocyte phagocytosis was examined by zymosan induced and luminol-aided chemiluminiscence assay in vitro. Results: The most important finding of this study was the immunosuppressing effect on leukocyte activity, hypolocomotion and increased behavioural response to modafinil-induced psychostimulation caused by prenatal exposure to the same drug. We did not detect significantly altered anxiety-related behaviour in any group disregarding the pre- and postnatal treatments. Conclusion: This is the first evidence of developmental toxicity of modafinil which needs to be taken into account as a potential risk factor when modafinil is administered to women who may become or are pregnant

Contemporary Czechoslovak Prints

The authors briefly outline the development of the exhibition and discuss printmaking in Czechoslovakia. Artists' statements. Biographical notes

Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

<div><p>Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H₂S) in the process of porcine oocyte aging. H₂S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H₂S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H₂S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H₂S from a donor (Na₂S.9H₂O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H₂S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H₂S on MPF and MAPK activities were detected and the intracellular mechanism underlying H₂S activity remains unclear, our study clearly demonstrates the role of H₂S in the regulation of porcine oocyte aging.</p></div

Dual Effects of Hydrogen Sulfide Donor on Meiosis and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes

<div><p>Hydrogen sulfide (H₂S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H₂S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H₂S donor, Na₂S, accelerated oocyte <i>in vitro</i> maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H₂S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H₂S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H₂S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.</p></div

Effect of H₂S donor on MPF and MAPK activity. 6A

<p>Histone H1 kinase assay was carried out to determine the activity of MPF by measurement of MPF capacity to phosphorylate its substrate (histone H1). <b>6B</b>: MBP kinase assay was carried out to determine the activity of MAPK by measurement of MAPK capacity to phosphorylate its substrate (MBP – Myelin basic protein). MPF and MAPK activities were determined in the MII oocytes (C – control, white column), the oocytes aged 12h and 24h in modified M199 medium, the oocytes aged 12h and 24h in modified M199 medium supplemented with a H₂S donor (Na₂S, black column), and the oocytes aged 12h and 24h in modified M199 medium supplemented with triple combination of inhibitors (3Ki, grey column). The results are presented as a ratio relative to the group of oocytes at metaphase II. <i>(GV – germinal vesicle stage; MII – oocytes at metaphase II; A12–12 hours of aging; A24–24 hours of aging; C – control, white column; Na₂S—Na₂S.9H₂O, 300 μM, black column; 3Ki - 1mM oxamic acid + 1mM beta-kyano-L-alanine + 5mM alpha-ketoglutaric acid disodium salt dihydrate). ^a,b, Statistically significant differences in activity (MPF or MAPK) between individual treatments at the same time are indicated with different superscripts (P<0.05)</i>.</p

Reversion of the effects of CBS, CSE and MPST inhibitors using a H₂S donor.

<p>Oocytes were cultivated to metaphase II and then exposed to prolonged cultivation (24 hours) in a modified M199 medium supplemented with a H₂S donor (Na₂S.9H₂O; 300 μM) and the following individual inhibitors: oxamic acid (1mM, OA), beta-kyano-L-alanine (1mM, KA), and alpha-ketoglutaric acid disodium salt dihydrate (5mM, KGA). <i>Na₂S (300 μM, Na₂S.9H₂O); KGA—alpha-ketoglutaric acid disodium salt dihydrate (5 mM); KA – beta-kyano-L-alanine (1mM); OA—oxamic acid (1 mM); MII—intact oocytes (oocytes at metaphase II, anaphase II or telophase II), A—activated oocytes (oocytes with pronuclei or embryos), F—fragmented oocytes, L—lysed oocytes; Different letters and numbers indicate significant differences between different treatments and hours of aging (P<0.05). A,B – statistically significant differences in portion of MII stage oocytes between individual treatments. a,b,c,d – statistically significant differences in portion of activated oocytes between individual treatments. 1,2,3 – statistically significant differences in portion of fragmented oocytes between individual treatments</i>.</p

Determination of endogenous H₂S production during porcine oocyte aging.

<p>Oocytes were cultivated to metaphase II (MII). Hydrogen sulfide production was carried out using the spectrophotometric method. MII oocytes, as well as oocytes exposed to prolonged cultivation for 24, 48 and 72 hours, were examined. The results of the measurement are presented as a ratio relative to the MII oocyte group. ^{<i>a, b</i>}<i>Statistically significant differences in spontaneous hydrogen sulfide production are indicated by different superscripts (P<0.05). Each experiment was repeated four times. The total number of oocytes in each sample was 100</i>.</p