In plants, posttranscriptional gene silencing (PTGS) is induced by small RNAs (sRNAs) generated from various dsRNA precursors. To assess the impact of dsRNA origin, we compared downregulation of GFP expression triggered by inverted repeat (IR), antisense (AS) and unterminated sense (UT) transcripts transiently expressed from the estradiol-inducible promoter. The use of homogeneously responding tobacco BY-2 cell lines allowed monitoring the onset of silencing and its reversibility. In this system, IR induced the strongest and fastest silencing accompanied by dense DNA methylation. At low induction, silencing in individual cells was binary (either strong or missing), suggesting that a certain threshold sRNA level had to be exceeded. The AS variant specifically showed a deviated sRNA-strand ratio shifted in favor of antisense orientation. In AS lines and weakly induced IR lines, only the silencer DNA was methylated, but the same target GFP sequence was not, showing that DNA methylation accompanying PTGS was influenced both by the level and origin of sRNAs, and possibly also by the epigenetic state of the locus. UT silencing appeared to be the least effective and resembled classical sense PTGS. The best responding UT lines behaved relatively heterogeneously possibly due to complexly arranged T-DNA insertions. Unlike IR and AS variants that fully restored GFP expression upon removal of the inducer, only partial reactivation was observed in some UT lines. Our results pointed out several not yet described phenomena and differences between the long-known silencer variants that may direct further research and affect selection of proper silencer variants for specific applications.

• Klíčová slova
• DNA methylation, PTGS, RNAi, Tobacco BY-2 cell line, dsRNA, siRNA,
• MeSH
• DNA bakterií MeSH
• dvouvláknová RNA genetika   MeSH
• metylace DNA MeSH
• posttranskripční úpravy RNA * MeSH
• regulace genové exprese u rostlin * MeSH
• reportérové geny MeSH
• RNA interference * MeSH
• RNA rostlin genetika   MeSH
• tabák genetika   MeSH
• umlčovací elementy transkripční MeSH
• umlčování genů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• dvouvláknová RNA MeSH
• RNA rostlin MeSH
• T-DNA MeSH Prohlížeč

The effect of DNA methylation upon posttranscriptional gene silencing (PTGS) has been investigated in transgenic tobacco lines showing PTGS and methylation of the neomycin phosphotransferase II (nptII) reporter genes. Application of the hypomethylation drugs dihydroxypropyladenine or 5-azacytidine resulted in approximately 30% reduced methylation of cytosines located in a non-symmetrical context in the 3' untranslated region of the nptII transgenes. The hypomethylation was accompanied by up to 12-fold increase in NPTII protein levels, suggesting that methylation of non-symmetrical motifs may account for an increased degree of PTGS. Models for the possible role of DNA methylation in PTGS are discussed.

• MeSH
• 3' nepřekládaná oblast genetika   MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• azacytidin farmakologie   MeSH
• cytosin metabolismus   MeSH
• DNA bakterií genetika   MeSH
• genetická transkripce účinky léků   genetika   MeSH
• geneticky modifikované rostliny MeSH
• jedovaté rostliny * MeSH
• kanamycinkinasa genetika   metabolismus   MeSH
• kultivované buňky MeSH
• metylace DNA účinky léků   MeSH
• otevřené čtecí rámce genetika   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• reportérové geny genetika   MeSH
• tabák cytologie   účinky léků   genetika   metabolismus   MeSH
• transgeny genetika   MeSH
• umlčování genů účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• 9-(2,3-dihydroxypropyl)adenine MeSH Prohlížeč
• adenin MeSH
• azacytidin MeSH
• cytosin MeSH
• DNA bakterií MeSH
• kanamycinkinasa MeSH
• T-DNA MeSH Prohlížeč

The aryl hydrocarbon receptor (AhR) transcription factor is activated by polycyclic aromatic hydrocarbons (PAH) and other ligands. Activated AhR binds to dioxin responsive elements (DRE) and initiates transcription of target genes, including the gene encoding prostaglandin endoperoxide synthase 2 (PTGS-2), which is also activated by the transcription factor NF-ĸB. PTGS-2 catalyzes the conversion of arachidonic acid (AA) into prostaglandins, thromboxanes or isoprostanes. 15-F2t-Isoprostane (IsoP), regarded as a universal marker of lipid peroxidation, is also induced by PAH exposure. We investigated the processes associated with lipid peroxidation in human alveolar basal epithelial cells (A549) exposed for 4 h or 24 h to model PAH (benzo[a]pyrene, BaP; 3-nitrobenzanthrone, 3-NBA) and organic extracts from ambient air particulate matter (EOM), collected in two seasons in a polluted locality. Both EOM induced the expression of CYP1A1 and CYP1B1; 24 h treatment significantly reduced PTGS-2 expression. IsoP levels decreased after both exposure periods, while the concentration of AA was not affected. The effects induced by BaP were similar to EOM except for increased IsoP levels after 4 h exposure and elevated AA concentration after 24 h treatment. In contrast, 3-NBA treatment did not induce CYP expression, had a weak effect on PTGS-2 expression, and, similar to BaP, induced IsoP levels after 4 h exposure and AA levels after 24 h treatment. All tested compounds induced the activity of NF-ĸB after the longer exposure period. In summary, our data suggest that EOM, and partly BaP, reduce lipid peroxidation by a mechanism that involves AhR-dependent inhibition of PTGS-2 expression. The effect of 3-NBA on IsoP levels is probably mediated by a different mechanism independent of AhR activation.

• Klíčová slova
• Aryl hydrocarbon receptor, Extractable organic matter, Lipid peroxidation, Polycyclic aromatic hydrocarbons,
• MeSH
• benz(a)anthraceny toxicita   MeSH
• benzopyren toxicita   MeSH
• buňky A549 MeSH
• cyklooxygenasa 1 metabolismus   MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• lidé MeSH
• mutageny toxicita   MeSH
• nádorové buněčné linie MeSH
• NF-kappa B metabolismus   MeSH
• peroxidace lipidů účinky léků   MeSH
• pevné částice toxicita   MeSH
• pneumocyty účinky léků   MeSH
• polycyklické aromatické uhlovodíky toxicita   MeSH
• receptory aromatických uhlovodíků metabolismus   MeSH
• transkripční faktory bHLH metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-nitrobenzanthrone MeSH Prohlížeč
• AHR protein, human MeSH Prohlížeč
• benz(a)anthraceny MeSH
• benzopyren MeSH
• cyklooxygenasa 1 MeSH
• cytochrom P-450 CYP1A1 MeSH
• mutageny MeSH
• NF-kappa B MeSH
• pevné částice MeSH
• polycyklické aromatické uhlovodíky MeSH
• receptory aromatických uhlovodíků MeSH
• transkripční faktory bHLH MeSH

It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele.

• MeSH
• alely MeSH
• epigeneze genetická MeSH
• genetická transkripce MeSH
• geneticky modifikované rostliny genetika   MeSH
• genomový imprinting MeSH
• malá interferující RNA genetika   MeSH
• metylace DNA * MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u rostlin * MeSH
• RNA interference MeSH
• tabák genetika   MeSH
• transgeny genetika   MeSH
• umlčovací elementy transkripční genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA MeSH

In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided.

• Klíčová slova
• DNA methylation, callus, dedifferentiation, histone modification, tobacco, transgene silencing,
• MeSH
• chromatin genetika   metabolismus   MeSH
• epigeneze genetická * MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• histony genetika   metabolismus   MeSH
• kostní svalek metabolismus   MeSH
• metylace DNA MeSH
• regulace genové exprese u rostlin MeSH
• tabák genetika   metabolismus   MeSH
• transgeny MeSH
• umlčování genů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• histony MeSH

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.

• MeSH
• alely * MeSH
• buněčné kultury MeSH
• epigeneze genetická * MeSH
• geneticky modifikované rostliny MeSH
• metylace DNA MeSH
• obrácené repetice genetika   MeSH
• otevřené čtecí rámce genetika   MeSH
• přeprogramování buněk genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regenerace genetika   MeSH
• regulace genové exprese u rostlin MeSH
• RNA rostlin metabolismus   MeSH
• sekvenční analýza DNA MeSH
• siřičitany MeSH
• Southernův blotting MeSH
• tabák genetika   fyziologie   MeSH
• transgeny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydrogen sulfite MeSH Prohlížeč
• RNA rostlin MeSH
• siřičitany MeSH

This article aimed to analyze concordance of parent- and child-reported child posttraumatic growth (PTG) following pediatric cancer, the influence of the parents' own level of PTG on the level of concordance and the influence of the parents' and the child's own level of PTG on the parents' proxy reports of PTG in the child. The sample included 127 parent-child dyads. The children provided self-reports of PTG and the parents provided reports of their own as well as the child's PTG. Overall, the results showed poor parent-child agreement on the child PTG, with the parents proxy-reporting higher levels of PTG than the children. The parents' proxy reports of the child PTG were the most accurate at the lowest levels of the parents' own level of PTG. The parents' own level of PTG was a stronger predictor of the parents' proxy reports than the child self-reported PTG. The results suggest that parents are not very accurate reporters of PTG in the child; therefore, their reports should be completed with child self-reports whenever possible.

• Klíčová slova
• benefit finding, childhood cancer survivors, parent–child concordance, posttraumatic growth,
• Publikační typ
• časopisecké články MeSH

Pediatric cancer can be considered an event potentially leading to posttraumatic stress symptoms (PTSS) as well as posttraumatic growth (PTG). While clinically significant levels of PTSS are rare in childhood cancer survivors, PTG is common in this population. However, the relationship of PTG to overall adaptation and quality of life (QOL) in pediatric cancer patients is not clear. Therefore, our study aims to analyse the relationships of PTSS and PTG with QOL in childhood cancer survivors. In this study, 172 childhood cancer survivors completed measures of quality of life (Minneapolis-Manchester Quality of Life Scale; child and adolescent version), posttraumatic stress (UCLA PTSD Reaction Index for DMS-IV) and posttraumatic growth (Benefit Finding Scale for Children). Correlation analyses were carried out separately for the child (up to 13 years, N = 47) and adolescent (more than 13 years, N = 125) groups and each QOL dimension. In the adolescent group, the relationship of PTSS and PTG with QOL was further verified by regression analyses while controlling for age, gender, and time off treatment. In children, negative relationships between PTSS and QOL were found, but the relationships between QOL and PTG were not significant. In adolescents, significant relationships were found for all dimensions of QOL and PTSS and also for several dimensions of QOL and PTG. The relationships between PTSS and QOL dimensions were negative in both groups, and the relationships between PTG and QOL in the adolescent group were weakly positive. In adolescents, regression analyses controlling for age, gender and time off treatment were performed and confirmed a negative relationship of PTSS with all QOL dimensions except for social functioning. For PTG, regression analyses revealed a significant positive relationship with QOL dimensions of social functioning, outlook on life and intimate relations. While the relationship between PTSS and QOL is negative for almost all QOL dimensions in children and adolescents, the nature of the relationship between PTG and QOL appears to be more complex and changing over time. PTG in children may reflect different processes with different outcomes than PTG in adolescents.

• Klíčová slova
• benefit finding, childhood cancer surivors, posttraumatic growth, posttraumatic stress, quality of life,
• Publikační typ
• časopisecké články MeSH

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

• Klíčová slova
• 5′ RNA degradome, Hop transformation, Lupulin biosynthesis, Plant promoter activation, Transcription factors, WRKY oligofamily,
• MeSH
• Humulus enzymologie   genetika   metabolismus   MeSH
• listy rostlin enzymologie   genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• terpeny * MeSH
• transkripční faktory genetika   metabolismus   MeSH
• umlčování genů fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lupulon MeSH Prohlížeč
• rostlinné proteiny MeSH
• terpeny * MeSH
• transkripční faktory MeSH

This theoretical article aims to summarize the results of studies relevant to parental influence on coping with childhood cancer and provide implications for future research focused on parent-child connections in posttraumatic growth (PTG) following childhood cancer. Parental influence on child coping described by the socialization of coping and socialization of emotions theories has already been studied in connection with posttraumatic stress, but the role of parents in the process of PTG in the child has not been clearly described yet. Several studies focused on PTG in childhood cancer survivors and their parents simultaneously, but only two studies explicitly included a parent-child connection in PTG in statistical analysis. Studies suggest that child PTG may be facilitated through parental coping advice supporting emotion expression and that parent-child connection in PTG may be mediated by the child's subjective perception of the parents' PTG. More research is needed to describe specific strategies proposed by parents and leading to child PTG and design tailored interventions for the use in the clinical care of childhood cancer survivors and their family.

• Klíčová slova
• benefit finding, parent–child relationship, pediatric cancer, posttraumatic growth (PTG), socialization of coping,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH