Cíl: Cílem práce je pojednání o transplantacích amniové membrány na Oční klinice Fakultní nemocnice Brno a retrospektivní zhodnocení souboru pacientů za období 2014–2019, kteří byli operováni pro různé indikace. Soubor a metodika: Retrospektivní vyhodnocení počtu a účinnosti jednotlivých druhů amniové membrány na souboru pacientů po transplantaci amniové membrány (AMT) pro různé indikace. Do sledovaného souboru bylo zařazeno celkem 134 pacientů, z toho 68 mužů a 66 žen. Medián věku byl 70 let. Celkový počet provedených transplantací amniové membrány během vybraných šesti let byl 139, přičemž rozložení v použití mražené (69 očí) a lyofilizované amniové membrány (70 očí) tvořilo polovinu. Technika AMT se volila na základě vstupního nálezu a diagnózy. Použitý typ amniové membrány (lyofilizovaná vs. mražená) závisel na urgentnosti výkonu. Výsledky: Na souboru 134 pacientů (139 očí) byl vyhodnocen počet transplantací amniových membrán a demonstrována jejich účinnost u jednotlivých diagnóz. Ve sledovací době bylo provedeno více transplantací amniových membrán za hospitalizace než v ambulantním režimu, a to u obou typů membrán (mražená i lyofilizovaná). Transplantace amniové membrány za hospitalizace byla provedena u 89 očí, v ambulantním režimu u 50 očí. Mezi indikace provedených transplantací amniové membrány patřila nejčastěji mikroperforace a perforace rohovky (30 očí), nehojící se defekty rohovky (21 očí), descemetokéla (19 očí), neurotrofické defekty (16 očí), ablace pterygia (12 očí) a lýza rohovky (11 očí). Ostatní stavy (léze spojivky, rekonstrukce fornixů, poleptání, periferní ulcerózní keratitida, oční jizevnatý pemfigoid a keratektomie) byly zastoupeny v menším počtu. Přes velmi rozmanitou skupinu indikací a pokročilý věk pacientů byla zjištěna velmi dobrá účinnost provedených transplantací amniové membrány. Někteří pacienti v průběhu sledovacího období zemřeli, limitací práce je tedy krátká a nestejná sledovací doba. Závěr: Úspěšnost AMT závisí nejen na správném načasování, indikaci a technice transplantace, ale i na compliance pacienta a dobře fungující spolupráci spádových očních lékařů.

Background: The aim of the work is a discussion of amniotic membrane transplantation at the Eye Clinic of the University Hospital Brno and a retrospective evaluation of a group of patients for the period 2014–2019 who were treated for various indications. Methods: Retrospective evaluation of the number and effectiveness of individual types of amniotic membrane in a group of patients after amniotic membrane transplantation (AMT) for various indications. A total of 134 patients were included in the study group, of which 68 were men and 66 were women. The median age was 70 years. The total number of amniotic membrane transplants performed during the selected six years was 139, with half the distribution using frozen (69 eyes) and lyophilized amniotic membrane (70 eyes). The AMT technique was chosen based on the initial finding and diagnosis. The type of amniotic membrane used (lyophilized vs. frozen) depended on the urgency of the procedure. Results: The number of amniotic membrane transplantations was evaluated in a group of 134 patients (139 eyes) and their effectiveness in individual diagnoses was demonstrated. During the follow-up period, more transplantations of amniotic membranes were performed during hospitalization than in the outpatient department, both types of membranes (frozen and lyophilized). Amniotic membrane transplantation during hospitalization was performed in 89 eyes, in the outpatient mode in 50 eyes. Indications for amniotic membrane transplantation included microperforation and corneal perforation (30 eyes), non-healing corneal defects (21 eyes), descemetocele (19 eyes), neurotrophic defects (16 eyes), ablation of pterygium (12 eyes) and corneal lysis (11 eyes). eyes). Other conditions (conjunctival lesions, fornix reconstruction, burns, peripheral ulcerative keratitis, ocular cicatricial pemphigoid and keratectomy) were represented in smaller numbers. Despite the very diverse group of indications and the advanced age of the patients, a very good efficacy of the performed amniotic membrane transplantations was found. Some patients died during the follow-up period, so the limitation of work is short and unequal follow-up period. Conclusion: The success of the procedure depends not only on the correct timing, indication and technique of transplantation, but also on patient compliance and well-functioning cooperation of regional ophthalmologists.

• MeSH
• amnion * transplantace   MeSH
• lidé MeSH
• oftalmologické chirurgické výkony metody   MeSH
• retrospektivní studie MeSH
• Check Tag
• lidé MeSH

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné jádro genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• epigeneze genetická MeSH
• FRAP MeSH
• G2 fáze MeSH
• histony metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• myši MeSH
• nika kmenových buněk MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• western blotting MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1β to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time τ = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to γH2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HPβ to UV-irradiated chromatin.

• MeSH
• acetylace MeSH
• buněčné linie MeSH
• buňky 3T3 MeSH
• časosběrné zobrazování MeSH
• chromatin metabolismus   účinky záření   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• FRAP MeSH
• histony metabolismus   MeSH
• inhibitory histondeacetylas metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kyseliny hydroxamové metabolismus   MeSH
• lidé MeSH
• myši MeSH
• poškození DNA MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.

• MeSH
• buněčné jadérko účinky léků   genetika   ultrastruktura   MeSH
• buněčné linie MeSH
• Cajalova tělíska účinky léků   genetika   ultrastruktura   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• daktinomycin farmakologie   MeSH
• fluorescenční barviva MeSH
• fluorescenční mikroskopie MeSH
• fotochemické procesy MeSH
• heterochromatin účinky léků   genetika   ultrastruktura   MeSH
• histony genetika   metabolismus   MeSH
• inhibitory histondeacetylas farmakologie   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• interfáze účinky léků   genetika   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lidé MeSH
• messenger RNA biosyntéza   MeSH
• mitóza účinky léků   genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Oct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• embryonální kmenové buňky cytologie   MeSH
• fibroblasty metabolismus   MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• histony chemie   MeSH
• kinetika MeSH
• myši MeSH
• oktamerní transkripční faktor 3 metabolismus   MeSH
• poškození DNA MeSH
• regenerativní lékařství metody   MeSH
• regulace genové exprese MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We have studied JMJD2b histone demethylase, which antagonizes H3K9me3 in the pericentromeric heterochromatin. In cells with a deficiency in the histone methyltransferase SUV39h, the level of full-length JMJD2b (JMJD2b-GFP-1086) at chromocenters was reduced, corresponding to a global decrease in JMJD2b and H3K9me3. In wild-type fibroblasts, the chromatin of ribosomal genes, which is dense with H3K9 methylation, lacked JMJD2b-GFP-1086, while mutant and truncated forms of JMJD2b densely occupied the nucleolar compartment. This implies that the PHD Zn-fingers and Tudor domains, which were removed in truncated JMJD2b, are responsible for the aberrant JMJD2b function. Intriguingly, the JMJD2b-GFP-1086 level was significantly higher in tumor cell nucleoli. The kinetic properties of JMJD2b-GFP-1086 in the nucleoli and nucleoplasm of normal and tumor cells were similar; ∼50% recovery of prebleached intensity was reached after <1 s. However, the mobile fraction of JMJD2b-GFP-1086 was increased in SUV39h-deficient cells. Similarly, the mobile fractions of mutant JMJD2b(1-424)H189A-GFP and truncated JMJD2b(1-424)GFP were greater than that measured for the full-length protein. We suggest that nucleoli are the site of an aberrant function of JMJD2b, the kinetic properties of which can be influenced by a mutant genetic background.

• MeSH
• buněčné jadérko genetika   metabolismus   MeSH
• buněčné linie MeSH
• chromatin metabolismus   MeSH
• chromatinová imunoprecipitace MeSH
• fibroblasty metabolismus   MeSH
• genová přestavba MeSH
• histonlysin-N-methyltransferasa genetika   MeSH
• Jumonjiho doména s histondemethylasami genetika   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• mutace MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• ribozomální DNA metabolismus   MeSH
• ribozomy metabolismus   MeSH
• zinkové prsty MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Here, we focus on epigenetic changes in leukaemia and MM (multiple myeloma) cells. We show how the histone signature, DNA methylation and levels of select tumour-suppressor proteins can be affected by inhibitors of HDACs (histone deacetylases) and Dnmts (DNA methyltransferases). Both inhibitors, TSA (trichostatin A) and 5-AZA (5-azacytidine), have the ability to change the histone signature in a tumour-specific manner. In MM cells, we observed changes in H3K4 methylation, while in leukaemia cells, H3K9 methylation was especially affected by select inhibitors. Compared with normal peripheral blood lymphocytes, tumour cell samples were characterized by increased H3K9 acetylation, increased H3K4me2, H3K9me2 and HP1α (heterochromatin protein 1α) levels and specific changes were also observed for DNA methylation. Additionally, we showed that the tumour suppressor pRb1 (retinoblastoma protein) is more sensitive to epigenetic-based anti-cancer stimuli than p53. We have found significant decrease in the levels of pRb1 and p53 in both myeloma and leukaemia cells after HDAC inhibition.

• MeSH
• antitumorózní látky farmakologie   MeSH
• azacytidin farmakologie   MeSH
• DNA modifikační methylasy genetika   metabolismus   MeSH
• epigeneze genetická MeSH
• histondeacetylasy metabolismus   MeSH
• histony genetika   metabolismus   MeSH
• inhibitory histondeacetylas farmakologie   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• leukemie farmakoterapie   genetika   MeSH
• lidé MeSH
• metylace DNA MeSH
• mnohočetný myelom farmakoterapie   genetika   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• retinoblastomový protein genetika   metabolismus   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

Telomeres are specialized chromatin structures that are situated at the end of linear chromosomes and play an important role in cell senescence and immortalization. Here, we investigated whether changes in histone signature influence the nuclear arrangement and positioning of telomeres. Analysis of mouse embryonic fibroblasts revealed that telomeres were organized into specific clusters that partially associated with centromeric clusters. This nuclear arrangement was influenced by deficiency of the histone methyltransferase SUV39h, LMNA deficiency, and the histone deacetylase inhibitor Trichostatin A (TSA). Similarly, nuclear radial distributions of telomeric clusters were preferentially influenced by TSA, which caused relocation of telomeres closer to the nuclear center. Telomeres also co-localized with promyelocytic leukemia bodies (PML). This association was increased by SUV39h deficiency and decreased by LMNA deficiency. These differences could be explained by differing levels of the telomerase subunit, TERT, in SUV39h- and LMNA-deficient fibroblasts. Taken together, our data show that SUV39h and A-type lamins likely play a key role in telomere maintenance and telomere nuclear architecture.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• epigeneze genetická MeSH
• fibroblasty metabolismus   MeSH
• genová přestavba MeSH
• intranukleární inkluzní tělíska metabolismus   MeSH
• lamin typ A metabolismus   MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• myši MeSH
• protein TRF1 metabolismus   MeSH
• průtoková cytometrie MeSH
• rap1 proteiny vázající GTP metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH