Nucleoside derivatives of 5-methylcytosine suppress 5-azacytidine-induced reactivation of a silent transgene in suspension-cultured tobacco cells.
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ABSTRACT:
SUBMITTER: Yamagishi K
PROVIDER: S-EPMC8215451 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN's) and adenosine 5'-phosphoramidate (NH2-pA) belonging to nucleoside 5'-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN's induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5'-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5'-phosphoramidates could be considered as new signaling molecules.
| S-EPMC8704414 | biostudies-literature
Project description:Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism.
| S-EPMC4235271 | biostudies-literature
Project description:Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti-inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow-2 (BY-2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 μm (mean ± SEM) amaranthin and 26.60 ± 1.53 μm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV-1 protease activity, whereas betanin did not.
| S-EPMC6587806 | biostudies-literature
Project description:Objectives/hypothesisVestibular neuritis is a common cause of both acute and chronic vestibular dysfunction. Multiple pathologies have been hypothesized to be the causative agent of vestibular neuritis; however, whether herpes simplex type I (HSV1) reactivation occurs within the vestibular ganglion has not been demonstrated previously by experimental evidence. We developed an in vitro system to study HSV1 infection of vestibular ganglion neurons (VGNs) using a cell culture model system.Study designbasic science study.ResultsLytic infection of cultured rat VGNs was observed following low viral multiplicity of infection (MOI). Inclusion of acyclovir suppressed lytic replication and allowed latency to be established. Upon removal of acyclovir, latent infection was confirmed with reverse-transcription polymerase chain reaction and by RNA fluorescent in situ hybridization for the latency-associated transcript (LAT). A total of 29% cells in latently infected cultures were LAT positive. The lytic ICP27 transcript was not detected by reverse-transcription polymerase chain reaction (RT-PCR). Reactivation of HSV1 occurred at a high frequency in latently infected cultures following treatment with trichostatin A (TSA), a histone deactylase inhibitor.ConclusionsVGNs can be both lytically and latently infected with HSV1. Furthermore, latently infected VGNs can be induced to reactivate using TSA. This demonstrates that reactivation of latent HSV1 infection in the vestibular ganglion can occur in a cell culture model, and suggests that reactivation of HSV1 infection a plausible etiologic mechanism of vestibular neuritis.
| S-EPMC3696486 | biostudies-literature
Project description:The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003). Journal Absract: (Plant Molecular Biology: 53, 2003) Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method. Experimenter name = Jim Murray Experimenter phone = 44 1223 334166 Experimenter fax = 44 1223 334162 Experimenter department = J Murray Laboratory Experimenter address = University of Cambridge Experimenter address = Institute of Biotechnology Experimenter address = Tennis Court Road Experimenter address = Cambridge Experimenter zip/postal_code = CB2 1QT Experimenter country = United Kingdom Keywords: time_series_design
2007-01-08 | GSE5750 | GEO
Project description:The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes.
| S-EPMC4117777 | biostudies-literature
Project description:An effective retarded suspension of ground granulated blast-furnace slag (GGBFS) needs a strong activator to reactivate the hydration. In this research study, sodium hydroxide (NaOH) as an alkali activator in two different concentrations (30 and 50 wt.%) was used to overcome the retardation and give the hardened GGBFS the reasonable strength. The study was carried out with a mixture of GGBFS, a solution of 1.0 wt.% d-gluconic acid (C₆H12O₇) as a retarder in the mixing water and a methyl cellulose as a stabilizer. The reactivation was executed after seven different periods (up to 28 days) after the system was retarded. The following investigations were performed: slump test, measurement of ultrasonic (US) velocity, compressive strength and gross density, thermogravimetry (TG) and scanning electron microscopy (SEM). The analyses of the hardened samples were carried out seven, 28 and 90 days after the reactivation. The result of the study is an effective reactivation of a retarded suspension. In this case, the activator with 50 wt.% NaOH shows a very high performance. The setting time of the reactivated binders is much longer compared to the reference, but, in the longer term, the compressive strength and the progress of the hydration exceed the performance of the reference.
| S-EPMC5456691 | biostudies-other
Project description:Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3' half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.
| S-EPMC3281821 | biostudies-literature
Project description:Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.
| S-EPMC8482155 | biostudies-literature
Project description:We encountered two cases of varicella occurring in newborn infants. Because the time between birth and the onset of the illness was much shorter than the varicella incubation period, the cases suggested that the infection was maternally acquired, despite the fact that neither mother experienced clinical zoster. Thus, we tested the hypothesis that VZV frequently reactivates asymptomatically in late pregnancy. The appearance of DNA-encoding VZV genes in saliva was used as an indicator of reactivation. Saliva was collected from 5 women in the first and 14 women in the third trimesters of pregnancy and analyzed at two different sites, at one using nested PCR and at the other using quantitative PCR (qPCR). No VZV DNA was detected at either site in the saliva of women during the first trimester; however, VZV DNA was detected in the majority of samples of saliva (11/12 examined by nested PCR; 7/10 examined by qPCR) during the third trimester. These observations suggest that VZV reactivation occurs commonly during the third trimester of pregnancy. It is possible that this phenomenon, which remains in most patients below the clinical threshold, provides an endogenous boost to immunity and, thus, is beneficial.
| S-EPMC9318610 | biostudies-literature