Cíl studie: Histony se vážou sekvenčně nespecifi ckým způsobem za vzniku chromatinu. N-koncováoblast histonů je cílovým místem posttranslačních modifi kací (fosforylace a acetylace). Předpokládáse, že tyto modifi kace regulují strukturu chromatinu, a tím mohou ovlivňovat transkripci,DNA replikaci, mitotickou aktivitu a reparaci DNA. Regenerace dlaždicobuněčného epitelu jeprovázena výraznou buněčnou atypií, pleomorfi smem jádra a jadérka a tyto změny mohou býtzaměněny s obrazem neoplazie. Cílem prezentované studie je detekce fosforylované a acetylovanéformy histonu H3 v cytologických stěrech a zhodnocení získaných výsledků ve vztahu k morfologickémuobrazu cervikální intraepiteliální neoplazie.Typ studie: Aplikovaný výzkum.Název a sídlo pracoviště: Základna experimentální onkologie, Masarykův onkologický ústav,Ústav patologické fyziologie a Klinika porodnictví a gynekologie, Masarykova univerzita v Brně.Metodika: Analyzovaný soubor tvořily cytologické stěry z děložního čípku 46 žen ve věku od 20 do46 let. 10 případů dlaždicobuněčné metaplazie, 20 CIN I, 12 CIN I, a 14 CIN III. Získané stěry bylypodrobeny imunohistochemické analýze s využitím polyklonálních protilátek proti fosforylovanéa acetylované formě histonu H3.Výsledky: Jaderná pozitivita pro fosforylované (P) a acetylované (A) formy histonu H3 byla vyššíu CIN II (23 % P, 33 % A) a CIN III (25 % P, 44 % A) ve srovnání s metaplazií (11 % P, 12 % A) a CIN I(8 % P, 15 % A).Závěr: Nalezli jsme souvislost mezi průkazem modifi kací histonu H3 a výskytem pokročilé formyCIN II a CIN III ve srovnání s CIN I a metaplazií. Barvení buněk s protilátkami rozlišujícími 1. fosforylovanou formu histonu H3 představuje vysoce specifi cký marker mitózy a 2. detekceacetylované formy koreluje s lokalizací transkripční aktivity.

Objective: Histones bind in a sequence-independent manner to form chromatin. The aminoterminaltails of histones are targets for both phosphorylation and acetylation events. These modifi cationsare thought to fundamentally regulate chromatin structure to accommodate transcription,DNA replication, mitosis and DNA repair. Regeneration of squamous epithelium is accompaniedby marked cellular atypia, nuclear and nucleolar pleomorphism which could be confused withneoplasia. The aim of the study was to detect phosphorylated and acetylated forms of histone H3in cytological smears.Design: Translational research.Setting: Department of Experimental Oncology, Masaryk Memorial Cancer Institute, Departmentof Pathological Physiology, Medical Faculty and Department of Obstetrics and Gynecology, MasarykUniversity, Czech Republic.Methods: Smears from women aged between 20 to 46 years were selected. The specimens comprised10 squamous metaplasia, 20 CIN I, 12 CIN II, and 14 CIN III. The smears were stained withpolyclonal antibodies against phosphorylated and acetylated forms of histone H3.Results: We found that nuclear positivity for phosphorylated (P) and acetylated (A) forms of histoneH3 in CIN II (23% P, 33% A) and CIN III (25% P, 44% A) was higher in comparison with CIN I(8% P, 15% A) and metaplasia (11% P, 12% A).Conclusion: We revealed a marked association of histone H3 modifi cations with the progressionof CIN II, CIN III in comparison with CIN I and metaplasia. Our results are in agreement withrecent fi ndings: 1. staining of cells with anti-phospho-histone H3 antibodies therefore provides ahighly specifi c marker for mitosis. 2. acetylation of nucleosomal histones correlates with localisedtranscriptional activity.

• MeSH
• acetylace MeSH
• cytodiagnostika metody   MeSH
• dospělí MeSH
• dysplazie děložního hrdla diagnóza   patologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• fosforylace MeSH
• histony fyziologie   MeSH
• imunohistochemie metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• přehledy MeSH
• srovnávací studie MeSH

Aurora kinase B (AURKB) is a chromosomal passenger protein that is essential for a number of processes during mitosis. Its activity is regulated by association with two other passenger proteins, INCENP and Survivin, and by phosphorylation on Thr 232. In this study, we examine expression and phosphorylation on Thr-232 of AURKB during meiotic maturation of pig oocytes in correlation with histone H3 phosphorylation and chromosome condensation. We show that histone H3 phosphorylation on Ser-10, but not on Ser-28, correlates with progressive chromosome condensation during oocyte maturation; Ser-10 phosphorylation starts around the time of the breakdown of the nuclear envelope, with the maximal activity in metaphase I, whereas Ser-28 phosphorylation does not significantly change in maturing oocytes. Treatment of oocytes with 50 microM butyrolactone I (BL-I), an inhibitor of cyclin-dependent kinases, or cycloheximide (10 microg/ml), inhibitor of proteosynthesis, results in a block of oocytes in the germinal vesicle stage, when nuclear membrane remains intact; however, condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm of BL-I- or cycloheximide-treated oocytes, respectively. In these treated oocytes, no or only very weak AURKB activity and phosphorylation of histone H3 on Ser-10 can be detected after 27 h of treatment, whereas phosphorylation on Ser-28 is not influenced. These results suggest that AURKB activity and Ser-10 phosphorylation of histone H3 are not required for chromosome condensation in pig oocytes, but might be required for further processing of chromosomes during meiosis.

• MeSH
• benzamidy farmakologie   MeSH
• chinazoliny farmakologie   MeSH
• chromozomy fyziologie   účinky léků   MeSH
• cykloheximid farmakologie   MeSH
• financování organizované MeSH
• fosforylace fyziologie   MeSH
• gama-butyrolakton analogy a deriváty   farmakologie   MeSH
• histony metabolismus   MeSH
• meióza fyziologie   MeSH
• oocyty růst a vývoj   MeSH
• prasata MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).

• MeSH
• apoptóza účinky záření   MeSH
• financování organizované MeSH
• fosforylace MeSH
• fytohemaglutininy farmakologie   MeSH
• histony chemie   metabolismus   MeSH
• lidé MeSH
• lymfocyty MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• poškození DNA MeSH
• techniky in vitro MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH

The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an indicator of received dose of gamma radiation after whole-body irradiation of rats. Wistar rats were irradiated by 1-10 Gy of gamma radiation by 60Co source. Value LD50/60 was 7.37 (4.68-8.05) Gy. Histone H2AX is phosphorylated by ATM kinase on serine 139 (γH2AX) quickly after the irradiation. It forms microscopically visible foci in the site of double strand breaks of DNA. Flow-cytometric method was used for quantitative detection. This study is the first one that evaluated dose-dependency of H2AX phosphorylation in peripheral lymphocytes of rats irradiated by whole-body dose 1-10 Gy. Our data show a dose-dependent increase in γH2AX in rat peripheral blood lymphocytes 1 h after whole-body irradiation by the dose of 1-10 Gy. We proved that phosphorylation of histone H2AX is a prompt and reliable indicator of the received radiation dose suitable for rapid measurement before the number of lymphocytes in peripheral blood starts to decrease. It can be used already 1 h after the irradiation for an estimation of the received dose of radiation. Blood samples can be stored in 4 °C for 23 h without significantly affecting the result.

• Klíčová slova
• histon H2AX,
• MeSH
• bioindikátory MeSH
• biologické markery MeSH
• celotělové ozáření MeSH
• dávka záření * MeSH
• fosforylace * MeSH
• histony * MeSH
• lymfocyty MeSH
• monitorování radiace * MeSH
• potkani Wistar MeSH
• průtoková cytometrie MeSH
• radiační expozice MeSH
• radiační účinky MeSH
• záření gama MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Lymphocytes are among the most radiosensitive cells. After exposure of the organism to ionizing radiation, they promptly die by apoptosis at a rate proportional to the dose received. Because of this, they are frequently used in biodosimetry. We demonstrated that one hour after whole-body irradiation of rats, histone H2AX in the lymphocyte nuclei was quickly phosphorylated on serine 139, the phosphorylation process being directly dependent on the gamma radiation dose. In the work presented here, we studied the kinetics of lymphocyte depletion in the peripheral blood and phosphorylation of histone H2AX in the peripheral blood lymphocytes after local (thoracic) irradiation of rats. Twenty-four hours after whole-body irradiation of the rats at a dose of 5 Gy, the lymphocyte count declined to almost zero values, whereas after local irradiation of the thorax area, the counts of lymphocytes in the peripheral blood remained unaltered. The authors employed two methods (flow-cytometric and microscopic) for the gammaH2AX determination in the peripheral blood lymphocytes, 1 h after thoracic irradiation of rats. Flow cytometry revealed a dose dependence on the increase in gammaH2AX in a dose range of 10-30 Gy. The microscopic method was more sensitive in the case of lower radiation doses, the dependence on the dose being obvious from a dose as low as 5 Gy. The methods are able, in the dose range 5-30 Gy, to differentiate between the type of irradiation, i.e. the whole-body or local.

• MeSH
• apoptóza účinky léků   účinky záření   MeSH
• buněčné jádro účinky záření   MeSH
• celotělové ozáření škodlivé účinky   MeSH
• financování organizované MeSH
• fosforylace účinky záření   MeSH
• histony účinky záření   MeSH
• hrudník cytologie   účinky záření   MeSH
• imunochemie metody   MeSH
• leukocyty mononukleární účinky záření   MeSH
• lymfocyty účinky léků   účinky záření   MeSH
• potkani Wistar MeSH
• průtoková cytometrie využití   MeSH
• radiometrie využití   MeSH
• statistika jako téma MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH

H2AX is a variant of histone H2A found in nuclei of eukaryotic cells. In 1998 it was found that after formation of double-strand breaks of DNA (DSB) due to ionizing radiation, H2AX is phosphorylated on serine 139 in the conserved COOH-terminal region. This phosphorylated form is termed ?H2AX. Further studies revealed that ?H2AX formation is an early event in DSB recognition and, subsequently, many proteins engaged in DNA repair, cell cycle regulation, chromatin remodeling and apoptosis are recruited to the DSB site. The ?H2AX presence is limited to the sites around DSB and the phosphorylation is proportional to the DNA damage extent. Methods such as Western blotting, flow cytometry and immunocytochemistry are used to detect ?H2AX. Detection of ?H2AX foci is useful to visualize localized DSB. Quantification of ?H2AX is useful for monitoring DNA damage. Further, ?H2AX is a potential molecular marker in aging and cancer. The present review covers current knowledge of the role of ?H2AX and of methods for its detection and quantification.

• MeSH
• dvouřetězcové zlomy DNA MeSH
• fosforylace MeSH
• histony MeSH
• poškození DNA MeSH
• průtoková cytometrie MeSH
• zlomy chromozomů MeSH

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.

• MeSH
• fosforylace účinky záření   MeSH
• G1 fáze účinky záření   MeSH
• G2 fáze účinky záření   MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• záření gama škodlivé účinky   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.

• MeSH
• acetylace MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• chromatin ultrastruktura   MeSH
• chromozomální proteiny, nehistonové fyziologie   MeSH
• epigeneze genetická MeSH
• exprese genu MeSH
• financování organizované MeSH
• histony genetika   metabolismus   MeSH
• interfáze MeSH
• lidé MeSH
• lidské chromozomy X metabolismus   MeSH
• metylace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH

BACKGROUND: Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level. RESULTS: Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase. CONCLUSIONS: By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

• MeSH
• Arabidopsis imunologie   MeSH
• chromatin fyziologie   MeSH
• flagelin imunologie   MeSH
• fosforylace MeSH
• fyziologický stres MeSH
• histondeacetylasy metabolismus   MeSH
• histony metabolismus   MeSH
• imunita rostlin * MeSH
• mitogenem aktivované proteinkinasy kinas metabolismus   MeSH
• přirozená imunita MeSH
• proteiny huseníčku metabolismus   MeSH
• restrukturace chromatinu * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The phosphorylation of histone H3 at Ser10, Ser28, Thr11 and Thr3 of the amino terminal has been proved related to mitosis of the mammalian cells. However, the function of the Thr3 phosphorylation of H3 remains unclear. In this study, indirect immunofluorescence labelling and laser confocal microscopy were used to examine the cellular dynamic distribution of Thr3-phosphorylated H3 at mitosis in CHO cells. The results showed that the Thr3 phosphorylation began at early prophase and spread throughout the chromosomes at late prophase. At metaphase, most of the Thr3-phosphorylated H3 was distributed along the entire chromosomal arms and maintained until early anaphase. During late anaphase and telophase, the fluorescent signal of Thr3-phosphorylated H3 disappeared from chromosomes. There was a precise spatial and temporal correlation between H3 phosphorylation of Thr3 and stages of chromatin condensation. The timing of Thr3 phosphorylation and dephosphorylation in mitosis were similar to that reported for Thr11 phosphorylation of H3. The Thr3-phosphorylated H3 localized along the arms of chromosomes during metaphase and early anaphase. It was different from the Ser10-phosphorylated H3, which localized at telomere regions, and Thr11-phosphorylated H3, which localized at centromeres. The results suggest that the Thr3 phosphorylation of histone H3 may play a specific role, which is different from Ser10 phosphorylation and Thr11 phosphorylation in mitosis.

• MeSH
• CHO buňky cytologie   účinky léků   MeSH
• chromatin genetika   MeSH
• financování vládou MeSH
• fluorescenční mikroskopie metody   využití   MeSH
• fosforylace fyziologie   MeSH
• histony genetika   účinky léků   MeSH
• mitóza fyziologie   genetika   MeSH
• western blotting metody   využití   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH