Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models

Antineoplastic Drugs as a Potential Risk Factor in Occupational Settings: Mechanisms of Action at the Cell Level, Genotoxic Effects, and Their Detection Using Different Biomarkers

U članku je prikazana osnovna podjela antineoplastičnih lijekova prema mehanizmima djelovanja na razini stanice. Objašnjeni su mehanizmi genotoksičnosti najvažnijih vrsta lijekova koji se primjenjuju u okviru uobičajenih protokola za liječenje zloćudnih novotvorina. Navedena je važeća klasifi kacija antineoplastika prema kancerogenom potencijalu, podaci o mutagenom potencijalu te je prikazana njihova podjela u skladu s anatomsko-terapijsko-kemijskim sustavom klasifi kacije. Sustavno su prikazani najvažniji rezultati svjetskih i hrvatskih istraživanja na populacijama radnika izloženih antineoplasticima, provedenih u razdoblju 1980.-2009. s pomoću četiri najčešće primjenjivane metode: analize izmjena sestrinskih kromatida, analize kromosomskih aberacija, mikronukleus-testa i komet-testa. Objašnjena su osnovna načela navedenih metoda te raspravljene njihove prednosti i nedostaci. Biološki pokazatelji daju važne podatke o individualnoj osjetljivosti profesionalno izloženih ispitanika koji mogu poslužiti unaprjeđenju postojećih uvjeta rada i upravljanju rizicima pri izloženosti genotoksičnim agensima. Na osnovi prednosti i nedostataka citogenetičkih metoda zaključeno je da je mikronukleus-test, koji podjednako uspješno dokazuje klastogene i aneugene učinke, jedna od najboljih metoda dostupnih za otkrivanje štetnih djelovanja antineoplastičnih lijekova koji su u aktivnoj primjeni.This article brings an overview of the mechanisms of action of antineoplastic drugs used in the clinical setting. It also describes the genotoxic potentials of the most important classes of antineoplastic drugs involved in standard chemotherapy protocols. Classifi cation of antineoplastic drugs according to the IARC monographs on the evaluation of carcinogenic risks to humans is accompanied by data on their mutagenicity and the most recent updates in the Anatomical Therapeutic Chemical (ATC) Classifi cation System. We report the main fi ndings of biomonitoring studies that were conducted in exposed healthcare workers all over the world between 1980 and 2009 using four biomarkers: sister chromatid exchanges, chromosome aberrations, micronuclei. and the comet assay. The methods are briefl y explained and their advantages and disadvantages discussed. Biomarkers provide important information on individual genome sensitivity, which eventually might help to improve current working practices and to manage the risks related with exposure to genotoxic agents. Taking into consideration all known advantages and drawbacks of the existing cytogenetic methods, the micronucleus assay, which is able to detect both clastogenic and aneugenic action, is the most suitable biomarker for assessing harmful effects of antineoplastic drugs currently used in health care

Modified neutral comet assay for human lymphocytes

Comet assay under neutral conditions allows the detection of DNA double-strand breaks, considered to be the biologically relevant radiation-induced lesion. In this report we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA double strand breaks in human lymphocytes irradiated with doses of 60Co gamma-rays (from 10 to 100 Gy). The analysis carried out according to this protocol takes less time than those published so far. Also, the use of lysis at 50°C is avoided; this is important in view of the presence of heat-labile sites in the DNA of irradiated cells, recently reported by Rydberg [12]. The comets have well defined, sharp limits, are suitable for computer image analysis and chromatin of the control cells remains condensed

Wsparcie inteligentnych specjalizacji w polskich regionach w ramach polityki spójności - przykład województwa lubelskiego

Subject and purpose of work: The aim of the work is to present the possibilities and assess the use of support for smart specializations under the EU cohesion policy 2014-2020 in the Lubelskie Voivodeship. Research tasks: classification of projects implemented under the ROP of the Lubelskie Voivodeship 2014-2020 (ROP LV) depending on their adaptation to the region's smart specializations; determining the size of support for the development of region's smart specializations. Materials and methods: The research material is a database of projects co-financed from EU funds for 2014-2020, projects implemented under axis 1 of ROP LV were analyzed. Results: The total value of funding for the region's smart specializations was PLN 194 million. The largest number of projects was implemented by enterprises within the specialization of IT and automation. About 20% of funds were acquired by entities from outside the smart specialization industries. Conclusions: ROP LV is an important tool to support research and development activities in the Lubelskie Voivodeship

DNA double-strand break rejoining in radioadapted human lymphocytes: evaluation by neutral comet assay and pulse-field gel electrophoresis

Adaptive response (AR), an enhanced resistance to a high dose of ionising radiation acquired after pretreatment with a very low dose, was estimated in normal human lymphocytes. The question posed was whether the extent of radioadaptation, assessed by micronucleus test, would be related to the rate of DNA double-strand break (DSB) rejoining. Phytohemagglutinin-stimulated G1-lymphocytes from 5 healthy male volunteers were pre-treated (or not) with an adaptive (5 cGy) dose of X-rays, followed by a higher (5 or 10 Gy) challenge dose after 20-22 h. DSB rejoining after the challenge dose was monitored with the use of two methods: neutral comet assay, modified to reduce the contribution of single-strand breaks (SSBs) and thermolabile sites, and pulse-field gel electrophoresis (PFGE), specific for DSBs. At the level of micronuclei, an AR was observed in lymphocytes of 3 of 5 donors. Up to 60 min, comet assay showed no statistically significant differences in DNA break rejoining between adapted and non-adapted lymphocytes, independently of AR appearance. PFGE gave similar results, although in three donors it revealed secondary increases in DSBs levels at 30 min and/or 60 min post-irradiation in the adapted vs. the non-adapted samples. Failure to demonstrate changes in DSBs rejoining rate in the adapted lymphocytes could be due to diversity of AR intensity/timing at the level of DNA repair in not fully homogenous cell populations. Also, “rare” DNA cuts characteristic of early apoptosis/necrosis could overlap the process of DNA break rejoining

Laserocarpum, a new genus of Apiaceae endemic to Greece

Laserpitium pseudomeum is an endemic umbellifer of Greece occurring in the mountains of Sterea Ellas and northern Peloponnese. Molecular data indicate that it is not related to its putative congeners, but instead constitutes an isolated lineage. The nuclear ribosomal DNA ITS tree places it as a weakly supported sister group to Portenschlagiella ramosissima, the sole species in its genus, whereas the tree inferred from three noncoding cpDNA loci does not confirm this relationship. Portenschlagiella ramosissima is sometimes placed in Athamanta, an affinity supported neither by molecular data nor by fruit morphology. Laserpitium pseudomeum notably differs from P. ramosissima in vegetative, flower, and fruit characteristics, and no obvious morphological synapomorphies indicate their close relationship. Therefore, we place L. pseudomeum in the newly described genus Laserocarpum and lectotypify the species with the specimen Orphanides 2019 (G00766460) at G-Boiss. © The Author(s) 2019

Effects of an antimutagen of 1,4-dihydropyridine series on cell survival and DNA damage in L5178Y murine sublines

In a series of studies it was shown that 1,4-dihydropyridine derivatives (1,4-DHP) show antimutagenic and anticlastogenic properties and accelerate repair of oxidant and ionising radiation generated DNA damage. Here, effects of one of 1,4-DHP compounds (sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate denoted as DHP) in X-irradiated L5178Y cells (murine lymphoma sublines, LY-R and LY-S) are reported. DHP treatment 1 h before, during and after X-irradiation gave a radioprotective effect in double strand break (DSB) repair competent LY-R cells: there was an increase in post-irradiation proliferation and cell viability as well as a slight acceleration of break rejoining as measured by the neutral comet assay. In the radiosensitive LY-S cells with impaired non-homologous end-joining system, the radioprotective effect was seen as enhanced growth and viability. There was, however, no effect on the DSB repair rate. Notably, there was no dependence of the biological effects on DHP concentration in the range of concentrations studied (1 nM - 100 mM), suggesting an all-or-none effect, as in cellular signaling induction observed in radioadaptation or bystander effect. We assume that DHP acts by decreasing fixation of radiation inflicted DNA damage, among others, by increasing the rate of DNA repair and enhancing the efficiency of checkpoint control. Direct confirmation of this assumption is necessary

DNA damage in subpopulations of human lymphocytes irradiated with doses in the range of 0-1 Gy of X-radiation

We compared three methods usually applied in biological dosimetry for estimation of radiation-induced DNA damage in human T and B lymphocytes: alkaline comet assay, micronucleus (MN) test and formation of histone gamma-H2AX foci. Human peripheral blood lymphocytes were fractionated using T cells and B cells isolation kits. Cells were irradiated with doses in the range of 0-1 Gy of X-rays. Induction of DNA damage was assessed by the standard alkaline comet assay, MN test and histone gammaH2AX foci immunofluorescence assay. Notwithstanding different end-points measured by the applied methods, all tests revealed a similar induction of DNA damage in B lymphocytes as compared with T lymphocytes. The results indicated that all three tests detect DNA damage with similar sensitivity, the lowest dose being approximately 0.3 Gy. The difference between irradiated and control cells was expressed as the ratio of the value obtained for irradiated cells (1 Gy) to that for control cells. The highest ratio was obtained for formation of gammaH2AX foci and was 6.2 for T and 13.8 for B lymphocytes, whereas those for comet assay and micronucleus test were 3.5; 3.6 and 5.6; 4.8, respectively