Efficiently Activated Serine Analogue is Not Transferred to Yeast tRNASer

Covalent attachment of cognate amino acid to the cognate tRNA is a prerequisite for the faithful synthesis of proteins in the cell. Aminoacylation of tRNA, catalyzed by aminoacyl-tRNA synthetases (aaRSs), proceeds by a two-step reaction whereby amino acid is first activated and then transferred to the 3\u27-ribose of tRNA. Serine hydroxamate (SerHX) is an interesting analogue of serine as it exhibits antimicrobial activity due to its inhibition of serylation in yeast and Escherichia coli. SerHX also mimics a noncognate substrate of yeast seryl-tRNA synthetase (ScSerRS) since it is efficiently activated and edited by this enzyme. However, whether this analogue is also transferred to tRNA during the second step of aminoacylation was not previously known. Here we show, for the first time, that aminoacylation of yeast tRNA with SerHX does not occur at a measurable rate, suggesting that the transfer is less tolerable toward SerHX than the activation step

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by “an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. Results: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Conclusions: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems

Antitumor effects of cyano-substituted organic compounds in vitro

U današnje vrijeme je potraga za novim kemoterapeuticima postala jedno od najvažnijih područja istraživanja unutar medicinske kemije. U ovom radu istražen je mehanizam rada dvaju novih cijano-supstituiranih heteroarila, koji citotoksično djeluju na staničnu liniju HeLa. Ovdje istraživani antitumorski spojevi su cijano-supstituirani naftotiofen (spoj 1) i cijano-supstituirani tieno-tiofen-karboksanilid (spoj 2). Za oba je spoja prethodno nađeno kako uzrokuju zastoj u fazi G1 i smrt stanica HeLa nakon tretmana u trajanju od 24 i 48 h. Mjerenjem aktivnosti kaspaze-3 pokazano je kako oba spoja povećavaju aktivnost tog enzima u stanicama HeLa nakon 48 h. Spoj 2 dodatno potiče apoptozu aktivacijom poli(ADP-riboza) polimeraza 1 (PARP-1). Kako bi razjasnili mehanizam kojim spojevi zaustavljaju stanični ciklus u fazi G1, metodom Western blot promatrana je razina ekspresije proteina regulatora staničnog ciklusa p53, p21 i p27. Ovime je pokazano kako oba spoja djeluju neovisno o p53. Spoj 1 povećava ekspresiju proteina p21 i p27, dok spoj 2 smanjuje njihovu ekspresiju. Fluorescencijskom mikroskopijom je nađeno kako oba spoja djeluju razorno na citoskeletne mreže tubulina i aktina, a analizom Western blot je utvrđeno da spoj 1 djeluje i na smanjenje njihove ekspresije. Spoj 1 pokazao je jače djelovanje na citoskeletni sustav. Mjerenjem proizvodnje reaktivnih kisikovih vrsta (ROS) ustanovljen je jači antioksidativni efekt spoja 2. Zajednički mehanizam djelovanja nije nađen, usprkos mnogim sličnostima među spojevima. Ovim rezultatima djelomično su rasvijetljeni mehanizmi protutumorskog djelovanja cijanosupstituiranih heteroarila na stanice HeLa.The discovery of novel potential chemotherapeutics has become one of the most important goals in medical chemistry. Here, mechanisms of actions are researched for two novel cyanoderivatives with a cytotoxic effect on HeLa cell line. These potent antitumor agents are cyanosubstituted naphtothiophene (compound 1) and cyano-substituted thieno-thiophenecarboxanilide (compound 2). It is has been previously reported that both compounds induce G1 arrest and apoptosis after 24h and 48h. Here, we measured an increase in activation of caspase-3 in HeLa cells after 48 h treatment for both compounds. Compound 2 additionally induced apoptosis by activation of poly(ADP-ribose) polymerase 1 (PARP-1). In order to elucidate the mechanism through which these compounds induce G1 arrest, Western blot analysis of cell cycle regulators, p53, p21 and p27, was performed. It was shown that both compounds act in a p53-independent manner. Compound 1 increases p21 and p27 expression, while compound 2 decreases expression of these proteins. The disruption of cytoskeletal networks of actin and tubulin for both compounds was established by fluorescence microscopy. Western blot analysis also showed deregulation of expression of cytoskeletal proteins by compound 1. Measurement of reactive oxygen species (ROS) formation showed stronger antioxidative effect of compound 2 on HeLa cells. Major mechanism of action, in spite of strong similarities, was not found. This research partially elucidated molecular aspects of action of cyano-substituted heteroaryls in HeLa cell line

Antitumor effects of cyano-substituted organic compounds in vitro

U današnje vrijeme je potraga za novim kemoterapeuticima postala jedno od najvažnijih područja istraživanja unutar medicinske kemije. U ovom radu istražen je mehanizam rada dvaju novih cijano-supstituiranih heteroarila, koji citotoksično djeluju na staničnu liniju HeLa. Ovdje istraživani antitumorski spojevi su cijano-supstituirani naftotiofen (spoj 1) i cijano-supstituirani tieno-tiofen-karboksanilid (spoj 2). Za oba je spoja prethodno nađeno kako uzrokuju zastoj u fazi G1 i smrt stanica HeLa nakon tretmana u trajanju od 24 i 48 h. Mjerenjem aktivnosti kaspaze-3 pokazano je kako oba spoja povećavaju aktivnost tog enzima u stanicama HeLa nakon 48 h. Spoj 2 dodatno potiče apoptozu aktivacijom poli(ADP-riboza) polimeraza 1 (PARP-1). Kako bi razjasnili mehanizam kojim spojevi zaustavljaju stanični ciklus u fazi G1, metodom Western blot promatrana je razina ekspresije proteina regulatora staničnog ciklusa p53, p21 i p27. Ovime je pokazano kako oba spoja djeluju neovisno o p53. Spoj 1 povećava ekspresiju proteina p21 i p27, dok spoj 2 smanjuje njihovu ekspresiju. Fluorescencijskom mikroskopijom je nađeno kako oba spoja djeluju razorno na citoskeletne mreže tubulina i aktina, a analizom Western blot je utvrđeno da spoj 1 djeluje i na smanjenje njihove ekspresije. Spoj 1 pokazao je jače djelovanje na citoskeletni sustav. Mjerenjem proizvodnje reaktivnih kisikovih vrsta (ROS) ustanovljen je jači antioksidativni efekt spoja 2. Zajednički mehanizam djelovanja nije nađen, usprkos mnogim sličnostima među spojevima. Ovim rezultatima djelomično su rasvijetljeni mehanizmi protutumorskog djelovanja cijanosupstituiranih heteroarila na stanice HeLa.The discovery of novel potential chemotherapeutics has become one of the most important goals in medical chemistry. Here, mechanisms of actions are researched for two novel cyanoderivatives with a cytotoxic effect on HeLa cell line. These potent antitumor agents are cyanosubstituted naphtothiophene (compound 1) and cyano-substituted thieno-thiophenecarboxanilide (compound 2). It is has been previously reported that both compounds induce G1 arrest and apoptosis after 24h and 48h. Here, we measured an increase in activation of caspase-3 in HeLa cells after 48 h treatment for both compounds. Compound 2 additionally induced apoptosis by activation of poly(ADP-ribose) polymerase 1 (PARP-1). In order to elucidate the mechanism through which these compounds induce G1 arrest, Western blot analysis of cell cycle regulators, p53, p21 and p27, was performed. It was shown that both compounds act in a p53-independent manner. Compound 1 increases p21 and p27 expression, while compound 2 decreases expression of these proteins. The disruption of cytoskeletal networks of actin and tubulin for both compounds was established by fluorescence microscopy. Western blot analysis also showed deregulation of expression of cytoskeletal proteins by compound 1. Measurement of reactive oxygen species (ROS) formation showed stronger antioxidative effect of compound 2 on HeLa cells. Major mechanism of action, in spite of strong similarities, was not found. This research partially elucidated molecular aspects of action of cyano-substituted heteroaryls in HeLa cell line

Nicotiana glauca Graham – potential cadmium phytoremediator

Zagađenje teškim metalima rezultat je ljudskog djelovanja, a njihova sve veća prisutnost ima ozbiljne posljedice na ekosustave, ali i na ljudsko zdravlje. Kadmij (Cd) je jedan od najtoksičnijih teških metala. Zbog svoje mobilnosti, akumulacije u hranidbenom lancu te široke primjene u industriji Cd predstavlja prijetnju za ljudsko zdravlje i okoliš u cjelini. Mnoge biljne vrste razvile su tolerantnost na teške metale u okolišu, koristeći dvije različite strategije: strategiju isključivanja ili strategiju akumulacije. Razumijevanje mehanizama akumulacije osobito je važno u prehrambeno važnim biljnim vrstama radi sprečavanja ulaska metala u hranidbeni lanac te radi moguće upotrebe biljaka za uklanjanje metala iz tla procesom fitoremedijacije. Cilj ovog istraživanja bio je usporediti mehanizme tolerancije na Cd u potencijalnom hiperakumulatoru vrsti Nicotiana glauca Graham i umjereno tolerantnoj vrsti N. tabacum L. Rezultati su pokazali kako je vrsta N. glauca hiperakumulator Cd te je kandidat za fitoremedijatora. U vrste N. glauca akumulacija i tolerancija Cd posredovana je povišenom konstitutivnom ekspresijom gena za unos i transport metala kroz biljku (ZIP, PDR3, HMA-A), gena za unos i održavanje homeostaze Fe (CIT1, FER), za antioksidativni odgovor (GST) te za sintezu helatora (NAS). Nakon tretmana s Cd u vrsti N. tabacum u listovima dolazi do odgovora sličnog deficijenciji Fe (povećanje ekspresije NRAMP1) te drastičnog smanjenja ekspresije transportera ZIP u korijenu, što dovodi do smanjenja unosa metala u biljku. U vrste N. glauca Cd uzrokuje povećanje ekspresije gena za unos metala u biljku i translokaciju metala u listove (HMA-A) što je važna karakteristika hiperakumulatora i potencijalnog fitoremedijatora. Dobiveni rezultati ukazuju da je upravo gen HMA-A potencijalna meta za konstrukciju transgenične vrste N. glauca koja bi se mogla koristiti za fitoremedijaciju Cd.Heavy metal pollution is a result of human actions, and their increasing presence has serious consequences for the environment, as well as for human health. Cadmium (Cd) is one of the most toxic metals. Because of its mobility, accumulation in the food chain and wide application in industry, Cd is a threat for human health and the entire environment. Many plant species have developed tolerance for heavy metals in the environment, by utilizing two distinct strategies: accumulation strategy and exclusion strategy. Understanding of accumulation mechanisms is especially important in agriculturally important crops in order to prevent heavy metals from entering the food chain and for possible use of plants for removal of metals from soils through the process of phytoremediation. The goal of this thesis was to compare mechanisms of tolerance to Cd in potential hyperaccumulator Nicotiana glauca Graham and moderately tolerant N. tabacum L. Results have shown that N. glauca is a Cd hyperaccumulator and a potential phytoremediator. In N. glauca Cd accumulation and tolerance is enabled by constitutively high expression of genes involved in heavy metal uptake and transport (ZIP, PDR3, HMA-A), Fe uptake and homeostasis (CIT1, FER), antioxidative defense (GST) and chelator synthesis (NAS). After Cd treatment in N. tabacum leaves there was a response similar to Fe-deficiency (induction of NRAMP1 expression) and drastic inhibition of ZIP transporter expression in the roots, in order to prevent further metals from entering the plant. In N. glauca Cd caused increased expression of gene involved in metal uptake and metal translocation to the leaves (HMA-A) which is an important trait of hyperaccumulators and potential phytoremediators. Obtained results show that gene HMA-A is a potential target for constructing transgenic N. glauca that could be used for Cd phytoremediation

Efficiently Activated Serine Analogue is Not Transferred to Yeast tRNASer

Covalent attachment of cognate amino acid to the cognate tRNA is a prerequisite for the faithful synthesis of proteins in the cell. Aminoacylation of tRNA, catalyzed by aminoacyl-tRNA synthetases (aaRSs), proceeds by a two-step reaction whereby amino acid is first activated and then transferred to the 3\u27-ribose of tRNA. Serine hydroxamate (SerHX) is an interesting analogue of serine as it exhibits antimicrobial activity due to its inhibition of serylation in yeast and Escherichia coli. SerHX also mimics a noncognate substrate of yeast seryl-tRNA synthetase (ScSerRS) since it is efficiently activated and edited by this enzyme. However, whether this analogue is also transferred to tRNA during the second step of aminoacylation was not previously known. Here we show, for the first time, that aminoacylation of yeast tRNA with SerHX does not occur at a measurable rate, suggesting that the transfer is less tolerable toward SerHX than the activation step