Early recruitment of PARP-dependent m8A RNA methylation at DNA lesions is subsequently accompanied by active DNA demethylation.

RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair.

Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells.

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

Irradiation by γ-rays reduces the level of H3S10 phosphorylation and weakens the G2 phase-dependent interaction between H3S10 phosphorylation and γH2AX.

Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph). On an individual cellular level, H3S10ph was low in highly γH2AX-positive UV laser-induced DNA lesions, and this nuclear distribution pattern was not changed by HDAC1 depletion. Despite this fact, spontaneous γH2AX-positive DNA lesions colocalized with large H3S10ph-positive nuclear bodies that appear in the G2 phase of the cell cycle. Similarly, by FLIM-FRET analysis, we observed an interaction between H3S10ph and γH2AX in the G2 phase. However, this interaction was reduced when cells were exposed to γ-rays. A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. Together, our data show that despite the fact that H3S10ph is not directly involved in DNA repair, a decrease in H3S10 phosphorylation and weakened interaction between H3S10ph and γH2AX is a result of radiation-induced damage of the genome. In this case, γ-irradiation also decreased the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX.

Depletion of A-type lamins and Lap2α reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2α protein is responsible for compactness of irradiated chromatin.

We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Mutations in the TP53 gene affected recruitment of 53BP1 protein to DNA lesions, but level of 53BP1 was stable after γ-irradiation that depleted MDC1 protein in specific TP53 mutants.

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

Function of heterochromatin protein 1 during DNA repair.

This review focuses on the function of heterochromatin protein HP1 in response to DNA damage. We specifically outline the regulatory mechanisms in which HP1 and its interacting partners are involved. HP1 protein subtypes (HP1α, HP1ß, and HP1γ) are the main components of constitutive heterochromatin, and HP1α and HP1ß in particular are responsible for heterochromatin maintenance. The recruitment of these proteins to DNA lesions is also important from the perspective of proper DNA repair mechanisms. For example, HP1α is necessary for the binding of the main DNA damage-related protein 53BP1 at DNA repair foci, which are positive not only for the HP1α protein but also for the RAD51 protein, a component of DNA repair machinery. The HP1ß protein also appears in monomeric form in DNA lesions together with the evolutionarily well-conserved protein called proliferating cell nuclear antigen (PCNA). The role of HP1 in DNA lesions is also mediated via the Kap1 transcription repressor. Taken together, these results indicate that the function of HP1 after DNA injury depends strongly on the kinetics of other DNA repair-related factors and their post-translational modifications, such as the phosphorylation of Kap-1.

PCNA is recruited to irradiated chromatin in late S-phase and is most pronounced in G2 phase of the cell cycle.

DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.

Advanced Image Acquisition and Analytical Techniques for Studies of Living Cells and Tissue Sections.

Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.

Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of γH2AX- and NBS1-positive repair foci.

BACKGROUND INFORMATION: The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. RESULTS: We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. CONCLUSION: We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most striking protein present not only at DNA lesions, but also spreading out in their vicinity. SIGNIFICANCE: Our conclusions highlight the significant role of the spatiotemporal dynamics of DNA repair-related proteins and their specific assembly/disassembly at DNA lesions, which can be cell type- and cell cycle dependent.

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle.

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies.

Cajal bodies are important nuclear structures containing proteins that preferentially regulate RNA-related metabolism. We investigated the cell-type specific nuclear distribution of Cajal bodies and the level of coilin, a protein of Cajal bodies, in non-irradiated and irradiated human tumor cell lines and embryonic stem (ES) cells. Cajal bodies were localized in different nuclear compartments, including DAPI-poor regions, in the proximity of chromocenters, and adjacent to nucleoli. The number of Cajal bodies per nucleus was cell cycle-dependent, with higher numbers occurring during G2 phase. Human ES cells contained a high coilin level in the nucleoplasm, but coilin-positive Cajal bodies were also identified in nuclei of mouse and human ES cells. Coilin, but not SMN, recognized UVA-induced DNA lesions, which was cell cycle-independent. Treatment with γ-radiation reduced the localized movement of Cajal bodies in many cell types and GFP-coilin fluorescence recovery after photobleaching was very fast in nucleoplasm in comparison with GFP-coilin recovery in DNA lesions. By contrast, nucleolus-localized coilin displayed very slow fluorescence recovery after photobleaching, which indicates very slow rates of protein diffusion, especially in nucleoli of mouse ES cells.

HP1ß-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs.

BACKGROUND: The repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways. RESULTS: We analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1ß (HP1ß) level. Moreover, Förster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1ß that was dependent on a functional chromo shadow domain of HP1ß. Thus, overexpression of HP1ß with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent. CONCLUSIONS: We propose that the simultaneous presence and interaction of UBF1 and HP1ß at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1ß. This might have functional significance for nucleotide excision repair.

Gene polymorphisms involved in manifestation of leucopenia, digestive intolerance, and pancreatitis in azathioprine-treated patients.

BACKGROUND: Approximately 10-28 % of patients experience adverse drug reactions related to treatment with thiopurines. The most serious reaction is myelosuppression, typically manifested as leucopenia, which occurs in approximately 2-5 % of patients. Other adverse drug reactions that often accompany thiopurine therapy are pancreatitis, hepatotoxicity, allergic reactions, digestive intolerance, arthralgia, febrile conditions, and rash. OBJECTIVE: The objective of this study was to assess the relationship between variant alleles of thiopurine S-methyltransferase (SNPs 238G > C, 460G > A and 719A > G), inosine triphosphate diphosphatase (SNPs 94C > A and IVS2 + 21A > C), and xanthine dehydrogenase (837C > T) and the occurrence of adverse drug reactions to azathioprine therapy. METHODS: Genotype was determined for 188 Caucasians diagnosed with inflammatory bowel disease treated with a standard dose of azathioprine (1.4-2.0 mg/kg/day). Allelic variants were determined by PCR-REA and real-time PCR methods. Results were statistically evaluated by use of Fisher's test and by odds ratio calculation. RESULTS: Variant genotype thiopurine S-methyltransferase predisposes to development of leucopenia (P = 0.003, OR = 5, CI 95 %, 1.8058-13.8444). Although not statistically significant, we observed a trend that suggested correlation between the occurrence of digestive intolerance and the variant genotype inosine triphosphate diphosphatase (P = 0.1102; OR 15.63, CI 95 %, 1.162-210.1094), and between the occurrence of pancreatitis and the variant allele xanthine dehydrogenase 837T (P = 0.1124; OR 12,1, CI 95 %, 1.15-126.37). CONCLUSION: The variant genotype thiopurine S-methyltransferase has been associated with the occurrence of leucopenia. The involvement of polymorphisms in inosine triphosphate diphosphatase and xanthine dehydrogenase genes in the development of digestive intolerance and pancreatitis will require further verification.

Separation and quantification of 9-(alkylthio)acridines by capillary micellar electrokinetic chromatography and capillary liquid chromatography.

Various thioacridine derivatives are potential chemotherapeutics against various diseases which are intensively synthesized, characterized, and investigated by many research groups. Efficient, fast, and reliable separation and quantification methods for their analysis are still to be developed. MEKC and capillary LC (CLC) were applied for the separation and quantification of five highly hydrophobic, weakly basic, and structurally similar 9-(alkylthio)acridines. Since the common anionic and cationic surfactants failed to separate the strongly hydrophobic thioacridines by MEKC, sodium cholate was used in an alkaline BGE and successfully employed for their fast separation. In CLC, the weakly basic nature of the thioacridines necessitated use of LiChrosorb RP-select B sorbent as the stationary phase, which combined with a very simple mobile phase methanol/water yielded an efficient chromatographic separation system. Both, the MEKC and CLC optimized separation methods were then applied to quantify the thioacridines within a concentration range of 1.0 x 10(-5)-1.0 x 10(-3) mol/L and the obtained experimental results were critically compared. In practical terms, the MEKC analytical method can quantify the analytes much faster but with a lower reliability while the CLC method performs slower analysis with a higher repeatability of the experimental results.

Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography.

Hydroxymethyl methacrylate-based monolithic columns for separation of oligonucleotides by capillary liquid chromatography (CLC) were prepared. We optimized composition of the polymerization mixture, which contained the monomer mixture consisting of N-(hydroxymethyl) methacrylamide (HMMAA) and ethylene dimethacrylate (EDMA), and the porogenic system composed of propane-1-ol, butane-1,4-diol and alpha, alpha'-azoisobutyronitrile (AIBN) as initiator. Separations of oligonucleotides were performed in HILIC (hydrophilic-interaction) mode using 100 mM triethylamine acetate (TEAA) in acetonitrile and in water as eluents. The influence of steepness of the mobile phase gradient on separation of the oligonucleotides was evaluated as well as the reproducibility of HMMAA monolith preparation.

Effects of oral alpha-tocopherol on lung response in rat model of allergic asthma.

OBJECTIVE AND BACKGROUND: Asthma is a chronic inflammatory disease in which an oxidant/antioxidant imbalance plays an important role. d-alpha-tocopherol (biologically the most active form of vitamin E) has redox properties and by scavenging the free radicals can act as an antioxidant. The aim of this study was to examine the effects of orally administered alpha-tocopherol in a rat model of allergic asthma. METHODOLOGY: Actively sensitized rats (OA) were treated with alpha-tocopherol (400 mg/kg/day for 10 days) or vehicle; 1 h after the last dose, they were challenged with antigen aerosol. The antigen-induced airway hyperresponsiveness to direct bronchoconstrictor (serotonin), the inflammatory cell infiltrate and histological changes were determined 1 or 24 h after the antigen challenge. RESULTS: Alpha-tocopherol pretreatment was not significantly effective at reducing the studied parameters when compared with controls, even though there was a tendency to a reduction in bronchial responsiveness and in eosinophil and neutrophil infiltration. CONCLUSION: Alpha-tocopherol when administered in the chosen study design in an animal model of asthma had no major effect on airway inflammation. The effect of antioxidants deserves further evaluation.

Influence of the spectrophotometric detection mode on the separation performance of systems with monolithic capillary columns: on-column and external-cell detection modes.

Poly(butyl methacrylate) monolithic columns were prepared by thermally initiated radical polymerization in fused-silica capillaries of 320 microm i.d. The prepared monolithic columns were tested by capillary liquid chromatography (CLC) combined with a UV-VIS spectrophotometric detector. The influence of the detection configuration (i.e., on-column and external-cell detection modes) on the performance of the chromatographic system was investigated. In the on-column detection mode within the monolith, the detection window was located inside the column section filled with the monolith. With the on-column detection configuration after the monolith, the detection window was positioned just behind the column section containing the monolith. Using the external-cell detection mode, an additional detection capillary, provided with a detection window defining the external-cell, was connected to the monolithic capillary column. These detection modes were critically compared in terms of the principal chromatographic parameters of the system involving the prepared monolithic capillary columns.

Monolithic organic polymeric columns for capillary liquid chromatography and electrochromatography.

This review briefly summarizes the present state of the preparation and use of capillary monolithic columns for liquid chromatography (LC) and electrochromatography (EC). Most important approaches to the preparation of monolithic stationary phases based on organic polymers are outlined and the properties of the monoliths obtained are compared with those of classical particulate phases. A few selected applications of monolithic columns are shown to demonstrate the most important advantages of monolithic capillary columns. It is concluded that both the monolithic and particulate capillary columns are important and that judicious choice of the type suitable for a particular application requires careful consideration of the purpose of the separation and the properties of the solutes to be separated. Monolithic columns are substantially younger than packed ones and thus will require further theoretical and experimental study to further improve their preparation and to enable reliable prediction of their properties and applicability; nevertheless, they are very promising for the future.