Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.     

Methods of assessment and clinical relevance of QT dynamics

The dependence on heart rate of the QT interval has been investigated for many years and several mathematical formulae have been proposed to describe the QT interval/heart rate (or QT interval/RR interval) relationship. While the most popular is Bazett's formula, it overcorrects the QT interval at high heart rates and under-corrects it at slow heart rates. This formulae and many others similar ones, do not accurately describe the natural behaviour of the QT interval. The QT interval/RR interval relationship is generally described as QT dynamics. In recent years, several methods of its assessment have been proposed, the most popular of which is linear regression. An increased steepness of the linear QT/RR slope correlates with the risk of arrhythmic death following myocardial infarction. It has also been demonstrated that the QT interval adapts to heart rate changes with a delay (QT hysteresis) and that QT dynamics parameters vary over time. New methods of QT dynamics assessment that take into account these phenomena have been proposed. Using these methods, changes in QT dynamics have been observed in patients with advanced heart failure, and during morning hours in patients with ischemic heart disease and history of cardiac arrest. The assessment of QT dynamics is a new and promising tool for identifying patients at increased risk of arrhythmic events and for studying the effect of drugs on ventricular repolarisation.     

Influence of system non-uniformity on dynamic phenomena in arrays of coupled nonlinear networks

In this paper we investigate the influence of system non-uniformity on the existence and stability of synchronous motion in an array of bi-directionally coupled electronic circuits. In computer simulations we find the level of non-uniformity for which synchronous behavior is sustained. We also present several examples of attractors, which appear when the synchronous motions is no longer stable.     

Effect of pH on the interfacial tension of bilayer lipid membrane formed from phosphatidylcholine or phosphatidylserine

The effect of pH of an electrolyte solution on the interfacial tension of lipid membrane formed from phosphatidylcholine (PC) or phosphatidylserine (PS) was studied. The relationships were well described by an equation presented earlier based on the Gibbs isotherm but only in the proximity of the isoelectric point. Therefore, in this work models have been derived to describe the adsorption of the H(+) and OH(-) ions at lipid surfaces formed from PC or PS, which would reproduce changes in interfacial tension more correctly, particularly in the ranges distant from the isoelectric point. In one model, the surface is continuous with uniformly distributed functional groups constituting the centres of H(+) and OH(-) ion adsorption while in the other the surface is built of lipid molecules, free or with attached H(+) and OH(-) ions. In both models, the contributions of the individual lipid molecule forms to the interfacial tension of the bilayer were assumed to be additive.     

Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis

BACKGROUND: Much attention is being paid to the biologic effects of magnetic fields (MFs). Although MFs enhance tumorigenesis, they are neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated. METHODS: To investigate the effect of MFs on apoptosis in HL-60 cells, we exposed the cells to static MFs of 6 mT generated by a magnetic disk of known intensity. Apoptosis was triggered by the DNA topoisomerase I inhibitor, camptothecin (CPT). Activation of caspases in situ using the fluorochrome-labeled inhibitor (FLICA) method and determination of plasma membrane integrity by excluding propidium iodide (PI) were measured by both laser scanning cytometry (LSC) and flow cytometry (FC). LSC and FC identified cells at three sequential stages of their demise: early apoptosis (cells with activated caspases and PI negative); late apoptosis (cells with activated caspases but unable to exclude PI); secondary necrosis (cells with apoptotic morphology no longer stained with FLICA, not excluding PI). RESULTS: MF alone did not induce any apoptogenic or necrogenic effect. CPT exposure led to the sequential appearance of apoptotic cells. In the presence of CPT and MF, the overall proportion of cells undergoing apoptosis was not significantly changed. However, we consistently observed a significant increase in the frequency of late apoptotic/necrotic cells when compared with samples treated with CPT alone (P < 0.001), as well as a decrease in the percentage of early apoptotic cells (P = 0.013). The data obtained by FC and LSC were consistent with each other, showing a similar phenomenon. CONCLUSION: Whereas MF alone or with CPT did not affect overall cell viability, it accelerated the rate of cell transition from apoptosis to secondary necrosis after induction of apoptosis by the DNA-damaging agent, CPT. Modulation of the kinetics of the transition from apoptosis to secondary necrosis by MF in vivo may play a role in inflammation and tumorigenesis.     

The immunosuppressive activity of peptide fragments of vaccinia virus C10L protein and a hypothesis on the role of this protein in the viral invasion

Our previous studies revealed that the 143-148 fragment of interleukin-1 receptor antagonist (IL-1 Ra) molecule with a Val-Thr-Lys-Phe-Tyr-Phe (VTKFYF) sequence inhibits the interleukin-1 (IL-1) interaction with its cellular receptor. The Val-Thr-Arg-Phe-Tyr-Phe (VTRFYF) sequence of the 322-327 fragment of the C-terminal domain of vaccinia virus protein related to the C10L vaccinia gene shows a very high homology to the 143-148 IL-1 Ra fragment, suggesting a similar inhibitory activity. To test this suggestion, we investigated the inhibitory activity of a series of synthetic peptides derived from 316 to 327 fragment of C10L on the interaction of IL-1 with its receptor. We also tested the peptides for their influence on the humoral and cellular immune response. The results indicate that biological activities of the C10L fragments are similar to those obtained for respective fragments of IL-1 Ra. The C-terminal domain of C10L protein can be easily folded into spatial structure similar to the crystallographic one of IL-1 Ra. Based on the crystallographic structure of IL-1 Ra, we constructed a 3-D model of the C10L protein. According to the model, the Val(322)-Asn(328) sequence is localized on the surface of the molecule and, therefore, it may be involved in the interactions with receptors. Our results indicate that the C10L viral protein can play an important role in vaccinia virus evasion of the host immune system. It may consist in the blockade of IL-1 receptors by the C10L protein, a homologue of the IL-1 Ra.     

Gas chromatographic determination of D-/L-arabinitol ratio in healthy Polish children

The D-/L-arabinitol enantiomers ratio (a marker of disseminated candidiasis of Candida species) in urine was determined by gas chromatography (GC) in 198 healthy Polish children ranging in age from 0 to 18 years. The urine samples were dry and trifluoroacetic anhydride (TFAA)-treated. Enantiomers derivatives were separated on a chiral column (beta-Dex 120, 60 m x 0.25 mm I.D.). A glass "solid-phase" injector and electron capture detector (ECD) were used. The ECD response was linear with correlation coefficients 0.999. The limit of detection was 0.02 micromol/l. Good results in terms of reproducibility, accuracy (RSD<10%, bias<6%), and linearity were obtained from real urine samples containing up to 400 micromol/l D-arabinitol. TFA-arabinitol derivatives in biological samples were stable from 1 to 5 days (depending on the arabinitol contents), while TFA-arabinitol standard derivatives were stable for 2 weeks. The identity of D- and L-arabinitol were confirmed by GC-MS analysis. The mean D-/L-arabinitol ratios ranged from 2.48 to 1.65 in the examined groups. The D-/L-arabinitol ratio was found to be exponentially regressive with age. A few cases of diagnosis of disseminated candidiasis by the GC method and confirmed by blood culture are described. The described GC method was also used for monitoring antifungal treatment of patients with disseminated candidiasis.     

Analysis of the microorganisms isolated from kidney recipients treated in the Department of Clinical Transplantology and General Surgery, Medical University in Bydgoszcz in 2001 year

One of the most important reasons of complications after organ transplantation may be the infections. The aim of the present work was to analyse of microorganisms isolated from patients, which were the recipients for kidney transplantation in 2001 year. The diagnostic material contained 140 samples from 53 patients, 40 (22.2%) samples from Euro-Collins fluid used for kidney storage before the transplantation and 3 end-pieces of catheter. The positive cultures were found in 125 (69.4%) samples. Gram-positive bacteria constituted 58.4%, Gram-negative bacteria--34.2%, fungi--7.4%. 140 strains of microorganisms were isolated from pharyngeal swabs and 55 strains of bacteria were isolated from palm swabs. Most of them were considered as a physiological flora. It was found 4-time significant bacteriuria among positive cultures from urine samples. In the cultures of fluid used for kidney storage 12 (30.0%) positive samples were obtained, out of which 16 strains of microorganisms were isolated. Among the strains of Staphylococcus 35.3% were MR. Among 18 strains of Gram-negative rods one strain was multiresistant to antibiotics. None of analyzed strains was ES beta L-producing. A high percentage of positive cultures from fluid used for kidney storage suggests the possibility of contamination of the organ with bacteria coming from kidney donor or during the storage, transport and actions connected with taking the organ to the transplantation.     

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.