Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication. The analysis also showed that very popular synchronisation protocols based on the double thymidine block can result in changes in the UNG2 content and uracil excision rate. In response, we propose a new method for the description of the changes of the content and the activity of different cell components during cell cycle without the necessity to use synchronisation protocols.
- MeSH
- Cell Nucleus metabolism MeSH
- Cell Cycle MeSH
- Kinetics MeSH
- Humans MeSH
- DNA Repair MeSH
- DNA Replication * MeSH
- S Phase MeSH
- Uracil-DNA Glycosidase * metabolism MeSH
- Uracil metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
... CONTENTS -- FOREWORD 9 -- ACKNOWLEDGEMENT 11 -- I. ... ... biomolecules 40 -- 3.1 Lipid peroxidation 40 -- 3.2 Oxidative damage to proteins 42 -- 3.3 Oxidative damage to DNA ... ... Oxidative stress 197 -- 2.2 Glycations 198 -- 2.3 Mitochondrial dysfunction 201 -- 2.4 Alteration of nuclear ... ... DNA 203 -- 2.5 Telomeres and cellular senescence 206 -- 2.6 Alteration of epigenome 209 -- 2.7 Collapse ...
Učební texty Univerzity Karlovy
First edition 241 stran : ilustrace ; 23 cm
- Conspectus
- Patologie. Klinická medicína
- Učební osnovy. Vyučovací předměty. Učebnice
- NML Fields
- biochemie
- patologie
- NML Publication type
- učebnice vysokých škol
BACKGROUND: Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. METHODS: 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. RESULTS: OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. CONCLUSION: In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.
- Publication type
- Journal Article MeSH
Oxidative stress and autophagy are potential mechanisms associated with cerebral ischemia/reperfusion injury (IRI) and is usually linked to inflammatory responses and apoptosis. Curcumin has recently been demonstrated to exhibit anti-inflammatory, anti-oxidant, anti-apoptotic and autophagy regulation properties. However, mechanism of curcumin on IRI-induced oxidative stress and autophagy remains not well understood. We evaluated the protective effects and potential mechanisms of curcumin on cerebral microvascular endothelial cells (bEnd.3) and neuronal cells (HT22) against oxygen glucose deprivation/reoxygenation (OGD/R) in vitro models that mimic in vivo cerebral IRI. The cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) activity assays revealed that curcumin attenuated the OGD/R-induced injury in a dose-specific manner. OGD/R induced elevated levels of inflammatory cytokines TNF-alpha, IL-6 as well as IL-1beta, and these effects were notably reduced by curcumin. OGD/R-mediated apoptosis was suppressed by curcumin via upregulating B-cell lymphoma-2 (Bcl-2) and downregulating Bcl-associated X (Bax), cleaved-caspase3 and TUNEL apoptosis marker. Additionally, curcumin increased superoxide dismutase (SOD) and glutathione (GSH), but suppressed malondialdehyde (MDA) and reactive oxygen species (ROS) content. Curcumin inhibited the levels of autophagic biomarkers such as LC3 II/LC3 I and Beclin1. Particularly, curcumin induced p62 accumulation and its interactions with keap1 and promoted NF-E2-related factor 2 (Nrf2) translocation to nucleus, accompanied by increased NADPH quinone dehydrogenase (Nqo1) and heme oxygenase 1 (HO-1). Treatment of curcumin increased phosphorylation-phosphatidylinositol 3 kinase (p-PI3K) and p-protein kinase B (p-AKT). The autophagy inhibitor 3-methyladenine (3-MA) activated the keap-1/Nrf2 and PI3K/AKT pathways. This study highlights the neuroprotective effects of curcumin on cerebral IRI.
- MeSH
- Antioxidants pharmacology metabolism MeSH
- Autophagy physiology MeSH
- Endothelial Cells metabolism MeSH
- NF-E2-Related Factor 2 metabolism MeSH
- Phosphatidylinositol 3-Kinases metabolism MeSH
- Kelch-Like ECH-Associated Protein 1 metabolism MeSH
- Curcumin * pharmacology MeSH
- Oxygen metabolism MeSH
- Neuroprotective Agents * pharmacology MeSH
- Oxidative Stress MeSH
- Proto-Oncogene Proteins c-akt metabolism MeSH
- Signal Transduction MeSH
- Publication type
- Journal Article MeSH
... CONTENTS -- Contents -- 1 Definition of the field and introduction to history (0. ... ... Slabý) 70 -- 5.1 Primary structure of nucleic acids 72 -- 5.2 Secondary structure of DNA 73 -- 5.2.1 ... ... Conformation of the DNA molecule 74 -- 5.2.2 Other non-canonical structures of the DNA molecule 75 -- ... ... Slabý) 86 -- 6.1 General aspects of DNA replication 86 -- 6.2 Function of DNA-polymerases 88 -- 6.3 Asymmetric ... ... double-strand breaks 168 -- 11.8.6 DNA damage tolerance 169 -- 12 Cellular communication (O. ...
1st edition 268 stran : ilustrace ; 30 cm
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- Učební osnovy. Vyučovací předměty. Učebnice
- NML Fields
- biologie
- NML Publication type
- učebnice vysokých škol
An endophytic fungus designated as EIT4T (MCC 9756T) was isolated from the asymptomatic stem tissue of Ephedra gerardiana collected from the Kargil district of Ladakh Union territory, India. Phylogenetic analysis based on concatenated nuclear ribosomal ITS (internal transcribed spacer) and LSU (large ribosomal subunit) sequence datasets revealed its placement within the genus Astragalicola. However, it formed a separate clade exhibiting strong bootstrap support value (80%). The highest nrITS sequence similarity between EIT4T and species of Astragalicola was 95.19% (A. vasilyevae) and 94.26% (A. amorpha), while nrLSU sequence similarity was 99.27% (A. amorpha). Morphologically, EIT4T differs from the other species of Astragalicola in having larger sub-globose to pyriform conidiomata, smaller and mostly unbranched conidiophores, and polymorphic translucent conidia with two terminal guttules. Based on combined cultural, micromorphological, molecular, and phylogenetic analyses, EIT4T represents a novel species in the genus Astragalicola proposed here as Astragalicola ephedrae sp. nov. Detailed description and illustrations of the novel species are provided. The type strain is EIT4T (= MCC 9756 T = MN29T).
- MeSH
- Ascomycota * MeSH
- DNA, Bacterial MeSH
- Ephedra * MeSH
- Phylogeny MeSH
- Fatty Acids analysis MeSH
- RNA, Ribosomal, 16S MeSH
- Sequence Analysis, DNA MeSH
- Bacterial Typing Techniques MeSH
- Base Composition MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- India MeSH
A critical aspect for obtaining accurate, reliable, and high-resolution estimates of nuclear DNA content is the release of nuclei from the cytoplasm in sufficient amounts, while maintaining their integrity throughout the analysis, protecting their DNA from degradation by endonucleases, and enabling stoichiometric DNA staining. In embryophytes, the most common method consists of chopping the plant material with a sharp razor blade to release nuclei into an isolation buffer, filtering the homogenate, and staining the nuclei in buffered suspension with a fluorochrome of choice. Despite the recent description of alternative approaches to isolate nuclei, the chopping procedure remains the most widely adopted method, due to its simplicity, rapidity, and effectiveness. In this review article, we discuss the specifics of nuclei isolation buffers and the distorting effects that secondary metabolites may have in nuclear suspensions and how to test them. We also present alternatives to the chopping procedure, options for filtering and fluorochromes, and discuss the applications of these varied approaches. A summary of the best practices regarding the isolation of plant nuclei for the estimation of nuclear DNA content is also provided.
Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.
- MeSH
- Cell Nucleus MeSH
- Ploidies MeSH
- Flow Cytometry MeSH
- Pollen * MeSH
- Spores * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.
- MeSH
- Acetylation MeSH
- Inclusion Bodies metabolism MeSH
- Cell Nucleolus metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cell Line MeSH
- Chromatin metabolism MeSH
- DNA metabolism MeSH
- Epigenesis, Genetic MeSH
- G-Quadruplexes * MeSH
- Transcription, Genetic * MeSH
- Histones metabolism MeSH
- Humans MeSH
- Methylation MeSH
- Mice MeSH
- DNA Repair MeSH
- Base Composition genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND AND AIMS: While nuclear DNA content variation and its phenotypic consequences have been well described for animals, vascular plants and macroalgae, much less about this topic is known regarding unicellular algae and protists in general. The dearth of data is especially pronounced when it comes to intraspecific genome size variation. This study attempts to investigate the extent of intraspecific variability in genome size and its adaptive consequences in a microalgal species. METHODS: Propidium iodide flow cytometry was used to estimate the absolute genome size of 131 strains (isolates) of the golden-brown alga Synura petersenii (Chrysophyceae, Stramenopiles), identified by identical internal transcribed spacer (ITS) rDNA barcodes. Cell size, growth rate and genomic GC content were further assessed on a sub-set of strains. Geographic location of 67 sampling sites across the Northern hemisphere was used to extract climatic database data and to evaluate the ecogeographical distribution of genome size diversity. KEY RESULTS: Genome size ranged continuously from 0.97 to 2.02 pg of DNA across the investigated strains. The genome size was positively associated with cell size and negatively associated with growth rate. Bioclim variables were not correlated with genome size variation. No clear trends in the geographical distribution of strains of a particular genome size were detected, and strains of different genome size occasionally coexisted at the same locality. Genomic GC content was significantly associated only with genome size via a quadratic relationship. CONCLUSIONS: Genome size variability in S. petersenii was probably triggered by an evolutionary mechanism operating via gradual changes in genome size accompanied by changes in genomic GC content, such as, for example, proliferation of transposable elements. The variation was reflected in cell size and relative growth rate, possibly with adaptive consequences.
