Tumor suppressor p53 is a key player in the cell response to DNA damage that suffers by frequent inactivating aberrations. Some of them disturb p53 oligomerization and influence cell decision between proliferation, growth arrest and apoptosis. Active p53 resides mostly in the nucleus, degradation occurs in the cytoplasm. Acute myeloid leukemia (AML)-related mutation of NPM (NPMmut) induces massive mislocalization of p53 to the cytoplasm, which might be related to leukemia initiation. Since both proteins interact and execute their function as oligomers, we investigated the role of perturbed p53 oligomerization in the p53 mislocalization process in live cells by FLIM (fluorescence lifetime imaging microscopy), fluorescence anisotropy imaging (FAIM), fluorescence cross-correlation spectroscopy (FCCS) and immunochemical methods. On a set of fluorescently labeled p53 variants, monomeric R337G and L344P, dimeric L344A, and multimeric D352G and A353S, we correlated their cellular localization, oligomerization and interaction with NPMmut. Interplay between nuclear export signal (NES) and nuclear localization signal (NLS) of p53 was investigated as well. While NLS was found critical for the nuclear p53 localization, NES plays less significant role. We observed cytoplasmic translocation only for multimeric A353S variant with sufficient stability and strong interaction with NPMmut. Less stable multimer D352G and L344A dimer were not translocated, monomeric p53 variants always resided in the nucleus independently of the presence of NPMmut and NES intactness. Oligomeric state of NPMmut is not required for p53 translocation, which happens also in the presence of the nonoligomerizing NPMmut variant. The prominent structural and functional role of the R337 residue is shown.

• MeSH
• akutní myeloidní leukemie * genetika   metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• jaderné lokalizační signály metabolismus   MeSH
• jaderné proteiny * genetika   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• mutace * MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 * metabolismus   genetika   chemie   MeSH
• nukleofosmin MeSH
• signály pro jaderný export MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

• MeSH
• apoptóza účinky léků   MeSH
• bisbenzimidazol chemie   MeSH
• buněčná smrt účinky léků   MeSH
• buněčné jádro účinky léků   metabolismus   MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• cisplatina farmakologie   MeSH
• fluorescenční mikroskopie metody   MeSH
• fluorescenční spektrometrie metody   MeSH
• fragmentace DNA účinky léků   MeSH
• kamptothecin farmakologie   MeSH
• lidé MeSH
• protinádorové látky farmakologie   MeSH
• průtoková cytometrie metody   MeSH
• reprodukovatelnost výsledků MeSH
• staurosporin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• barvení a značení MeSH
• buněčné jádro * genetika   MeSH
• DNA rostlinná genetika   MeSH
• ploidie * MeSH
• průtoková cytometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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
• buněčné jádro MeSH
• ploidie MeSH
• průtoková cytometrie MeSH
• pyl * MeSH
• spory * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Microalgae are photosynthetic microorganisms with a major influence on global ecosystems. Further, owing to the production of various secondary metabolites, microalgae are also intensively studied for their enormous potential in biotechnology and its applications. While flow cytometry (FCM) is a fast and reliable method particularly suitable for genome size estimation in plant and animal studies, its application to microalgae often comes with many methodological challenges due to specific issues (e.g., cell wall composition, and presence of various secondary metabolites). Sample preparation requires considerable amounts of biomass, chemical fixation, and/or extraction of cellular components. In genome size estimation, appropriate methods for isolation of intact nuclei (using lysis buffers, razor-blade chopping, various enzymes, or bead-beating of cells) are essential for successful and high-quality analyses. Nuclear DNA amounts of microalgae diverge greatly, varying by almost 30,000-fold (0.01 to 286 pg). Even though new algal reference standards for genome size are now being introduced, animal red blood cells and nuclei from plant tissues are still predominantly used. Due to our limited knowledge of microalgal life cycles, particular caution should be taken during 1C/2C-value (or ploidy level) assignments.

• MeSH
• biomasa MeSH
• biotechnologie MeSH
• délka genomu MeSH
• ekosystém MeSH
• mikrořasy * MeSH
• průtoková cytometrie MeSH
• Publikační typ
• časopisecké články MeSH

Fibrates belong to a group of ligands of peroxisome proliferator-activated receptor alpha (PPARα), which play a role in the regulation of CYP epoxygenases and soluble epoxide hydrolase (sEH), key enzymes in the metabolism of biologically highly active epoxyeicosatrienoic acids (EETs). We demonstrated that low doses of fibrates stimulate proliferation of the MCF7 cell line, while high doses suppress it. The increase in cell proliferation was accompanied by an increase in CYP epoxygenases and decrease in sEH levels. The overall level of PPARα remained same after low-dose fibrate stimulation; however, there was a significant shift of the receptor to the cell nucleus. PPARα expression was further demonstrated by immunohistochemistry in both carcinoma and healthy breast tissue samples both in the cytoplasm and in the nuclei. We have also observed higher nuclear PPARα positivity in tumor tissues. Although our results obtained for MCF7 cells suggest the potential role of PPARα in cell proliferation, we did not find an association between nuclear localization of PPARα and the expression of proliferation marker Ki-67 in tumor tissues. The exact role of PPARα in carcinogenesis still remains unclear.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• deriváty kyseliny fibrové farmakologie   MeSH
• epoxid hydrolasy metabolismus   MeSH
• geny erbB-2 * MeSH
• inhibiční koncentrace 50 MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory prsu metabolismus   patologie   MeSH
• PPAR alfa metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• pyrimidiny farmakologie   MeSH
• receptory pro estrogeny metabolismus   MeSH
• receptory progesteronu metabolismus   MeSH
• rozpustnost MeSH
• subcelulární frakce metabolismus   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• transport proteinů účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation. RESULTS: To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1). CONCLUSIONS: Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.

• MeSH
• buněčné jádro metabolismus   MeSH
• HeLa buňky MeSH
• helikasy RecQ genetika   MeSH
• helikáza Wernerova syndromu metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• metabolické sítě a dráhy fyziologie   MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   MeSH
• oxidace-redukce MeSH
• posttranskripční úpravy RNA fyziologie   MeSH
• proliferace buněk fyziologie   MeSH
• proteiny vázající RNA metabolismus   MeSH
• transport RNA fyziologie   MeSH
• Wernerův syndrom metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

• MeSH
• buněčný cyklus fyziologie   MeSH
• časové faktory MeSH
• chromozomy rostlin * metabolismus   MeSH
• genom rostlinný genetika   MeSH
• genomika metody   MeSH
• hydroxymočovina MeSH
• metafáze MeSH
• mitotický index MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH
• průtoková cytometrie metody   MeSH
• pufry MeSH
• Saccharum cytologie   genetika   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The standard therapy for malignant primary bone tumors such as osteosarcoma involves major surgeries. For tumors located in difficult regions such as the pelvis, surgical intervention could lead to serious side effects for example loss of a limb and/or function, loss of bowel, bladder and sexual function as well as problems with wound healing and surgical complications. Therefore, exploring other approaches that can improve or complement current surgical techniques is important. Hence, sensitizing primary bone tumors to radiation could offer an additional strategy that could complement surgery and significantly improve survival and quality of life. Gold nanoparticles (AuNPs) have been shown to enhance radiosensitivity by increasing the local dose of radiation inside tumors. Therefore, the referred procedure of preparation and functionalization of gold nanoparticles may be used for investigation whether DNA repair inhibition in the presence of AuNPs leads to an effective radiosensitizing strategy for primary bone tumor cells and explore the mechanism of how this may be happening. In our work, we prepared gold nanoparticles and verified the relation between the size of the AuNPs and their uptake in tumor 143B cells and also investigated whether the optimal size of the AuNPs should not be smaller than the size of nuclear envelope pores (20-50 nm). Hence, two different AuNPs systems were prepared: the first one with AuNPs core size of about 5 nm (BS) and the second one with AuNPs core size of about 50 nm (ZA). For cellular AuNPs uptake enhancement, we functionalized the AuNPs with signaling peptides. For this purpose we prepared PEG-coated AuNPs functionalized with signal peptides for targeted transport into the cytoplasm (CPP) and into the cell nucleus (CPP + NLS). The toxicity of the AuNPs systems was assessed by MTS assay. We prepared stable functionalized AuNPs systems of both sizes. With the functionalizing of the AuNPs using signal peptides (CPP, NLS), the AuNPs penetrated into the cell nucleus. The referred procedure of preparation and functionalization of gold nanoparticles may be used for investigating inhibition of DNA repair in the presence of AuNPs and it could lead to new understanding in overcoming radioresistance in primary bone tumor cells.

• MeSH
• buněčné jádro MeSH
• intracelulární signální peptidy a proteiny * MeSH
• kovové nanočástice * MeSH
• kvalita života MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nosiče léků * MeSH
• osteosarkom * MeSH
• radiosenzibilizující látky farmakologie   MeSH
• transport proteinů MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In-cell NMR spectroscopy is a unique tool that enables the study of the structure and dynamics of biomolecules as well as their interactions in the complex environment of living cells at near-to-atomic resolution. In this article, detailed instructions are described for setting up an in-cell NMR experiment for monitoring structures of DNA oligonucleotides introduced into nuclei of living human cells via tailored electroporation. Detailed step-by-step protocols for both the preparation of an in-cell NMR sample as well as protocols for conducting essential control experiments including flow cytometry and confocal microscopy are described. The strengths and limitations of in-cell NMR experiments are discussed. © 2018 by John Wiley & Sons, Inc.

• MeSH
• buněčné jádro metabolismus   MeSH
• cytosol metabolismus   MeSH
• elektroporace MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• nukleové kyseliny metabolismus   MeSH
• průtoková cytometrie MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH