A new technique for the analysis of metabolic pathways of cytidine analogues and cytidine deaminase activities in cells.

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation.

Basic Methods of Cell Cycle Analysis.

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.

Triterpenoid pyrazines and pyridines - Synthesis, cytotoxicity, mechanism of action, preparation of prodrugs.

A set of fifteen triterpenoid pyrazines and pyridines was prepared from parent triterpenoid 3-oxoderivatives (betulonic acid, dihydrobetulonic acid, oleanonic acid, moronic acid, ursonic acid, heterobetulonic acid, and allobetulone). Cytotoxicity of all compounds was tested in eight cancer and two non-cancer cell lines. Evaluation of the structure-activity relationships revealed that the triterpenoid core determined whether the final molecule is active or not, while the heterocycle is able to increase the activity and modulate the specificity.

Anchored linear oligonucleotides: the effective tool for the real-time measurement of uracil DNA glycosylase activity.

Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Förster resonance energy transfer.

Strengths and Weaknesses of Cell Synchronization Protocols Based on Inhibition of DNA Synthesis.

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages.

DNA Dyes-Highly Sensitive Reporters of Cell Quantification: Comparison with Other Cell Quantification Methods.

Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available.

New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis.

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation.

A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy.

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas' DNA by DAPI or Hoechst dyes.

Quantification of fixed adherent cells using a strong enhancer of the fluorescence of DNA dyes.

Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here.

DNA Replication: From Radioisotopes to Click Chemistry.

The replication of nuclear and mitochondrial DNA are basic processes assuring the doubling of the genetic information of eukaryotic cells. In research of the basic principles of DNA replication, and also in the studies focused on the cell cycle, an important role is played by artificially-prepared nucleoside and nucleotide analogues that serve as markers of newly synthesized DNA. These analogues are incorporated into the DNA during DNA replication, and are subsequently visualized.

Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2'-deoxyuridine, low concentration of hydrochloric acid and exonuclease III.

The approach for the detection of replicational activity in cells using 5-bromo-2'-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III.

Looking for ugly ducklings: The role of the stability of BrdU-antibody complex and the improved method of the detection of DNA replication.

5-Bromo-2'-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation.

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine.

5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines.

Tracking Mitochondrial DNA In Situ.

The methods of the detection of (1) non-labeled and (2) BrdU-labeled mitochondrial DNA (mtDNA) are described. They are based on the production of singlet oxygen by monovalent copper ions and the subsequent induction of DNA gaps. The ends of interrupted DNA serve as origins for the labeling of mtDNA by DNA polymerase I or they are utilized by exonuclease that degrades DNA strands, unmasking BrdU in BrdU-labeled DNA.

The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions.

We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells.

A fatal combination: a thymidylate synthase inhibitor with DNA damaging activity.

2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role.

Tracking DNA and RNA sequences at high resolution.

In this chapter, the basic principles and protocols of the electron microscopical detections of specific DNA and RNA sequences are described. We focused primarily on a comparison of various methods of electron microscopy in situ hybridization (EM-ISH) with respect to their sensitivity and the structural preservation of the sample with the aim of helping the readers select the appropriate hybridization protocol. As the post-embedding EM-ISH most frequently represents the optimal choice, the protocol for the post-embedding EM-ISH approach is described in detail.

A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA.

The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA.

Atomic scissors: a new method of tracking the 5-bromo-2'-deoxyuridine-labeled DNA in situ.

A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.

Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine -- the method for the effective suppression of this cross-reactivity.

5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU.

Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations.

Background: Cercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen Schistosoma mansoni.

In situ reverse transcription: the magic of strength and anonymity.

In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2'-deoxyuridine into the growing cDNA strand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin, 5-bromo-2'-deoxyuridine is 'invisible' in the DNA-DNA duplex but easily detectable in the DNA-RNA duplex.

Organization of human replicon: singles or zipping couples?

According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA.

Pontin is localized in nucleolar fibrillar centers.

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I.

Positioning of NORs and NOR-bearing chromosomes in relation to nucleoli.

It is widely accepted that chromosomes occupy more or less fixed positions in mammalian interphase nucleus. However, relation between large-scale order of chromosome positioning and gene activity remains unclear. We used the model of the human ribosomal genes to address specific aspects of this problem.