Three series of isomeric pyrrolo-and furo-fused 7deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine and furo[2′,3′:4,5]pyrrolo-[2,3-d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3′,2′,4,5]pyrrolo[2,3-d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide's ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) exhibit multiple effects on cell death pathways in mammalian cells. Thus, they are able to induce apoptosis by itself or promote cell death induced by other cytotoxic stimuli [King et al., 2004; Murn et al., 2004]. On the other hand, TLCK and TPCK were reported to prevent apoptosis by inhibiting the processing of caspases in response to some cell death inducing stimuli [Stefanis et al., 1997; Jones et al., 1998]. We observed that the pretreatment of HL-60 cells with TLCK or TPCK diminished caspases 3 and -7 (DEVDase) and caspase-6 (VEIDase) activity in response to various cell death inducing stimuli such as staurosporine (STS), etoposide (ETP), or N6-(2-isopentenyl)adenosine. In addition, TLCK but not TPCK inhibited collapse of mitochondrial transmembrane potential Delta Psi m (delta psi) in dying HL-60 cells. Such effects used to be considered as protective, however, the protection was only presumable since neither TLCK nor TPCK actually prevented cells from death. Our results further indicated that serine protease inhibitors TLCK and particularly TPCK acted as efficient direct inhibitors of mature caspases. Indeed, experiments with human recombinant caspases provided unequivocal evidence that TLCK and TPCK are very potent but non-specific inhibitors of activated caspases, namely caspases 3, -6, and -7. Interestingly, TPCK exhibited similar efficiency towards human recombinant caspases to that found for panspecific caspase inhibitor Boc-D-CMK. Such properties of TLCK and TPCK, previously considered as specific inhibitors of serine proteases, might offer novel consistent explanation for several protective or protective-like effects on apoptotic cells.

Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.

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