BACKGROUND: Silene vulgaris (bladder campion) is a gynodioecious species existing as two genders - male-sterile females and hermaphrodites. Cytoplasmic male sterility (CMS) is generally encoded by mitochondrial genes, which interact with nuclear fertility restorer genes. Mitochondrial genomes of this species vary in DNA sequence, gene order and gene content. Multiple CMS genes are expected to exist in S. vulgaris, but little is known about their molecular identity. RESULTS: We assembled the complete mitochondrial genome from the haplotype KRA of S. vulgaris. It consists of five chromosomes, two of which recombine with each other. Two small non-recombining chromosomes exist in linear, supercoiled and relaxed circle forms. We compared the mitochondrial transcriptomes from females and hermaphrodites and confirmed the differentially expressed chimeric gene bobt as the strongest CMS candidate gene in S. vulgaris KRA. The chimeric gene bobt is co-transcribed with the Cytochrome b (cob) gene in some genomic configurations. The co-transcription of a CMS factor with an essential gene may constrain transcription inhibition as a mechanism for fertility restoration because of the need to maintain appropriate production of the necessary protein. Homologous recombination places the gene cob outside the control of bobt, which allows for the suppression of the CMS gene by the fertility restorer genes. We found the loss of three editing sites in the KRA mitochondrial genome and identified four sites with highly distinct editing rates between KRA and another S. vulgaris haplotypes (KOV). Three of these highly differentially edited sites were located in the transport membrane protein B (mttB) gene. They resulted in differences in MttB protein sequences between haplotypes. CONCLUSIONS: Frequent homologous recombination events that are widespread in plant mitochondrial genomes may change chromosomal configurations and also the control of gene transcription including CMS gene expression. Posttranscriptional processes, e.g. RNA editing shall be evaluated in evolutionary and co-evolutionary studies of mitochondrial genes, because they may change protein composition despite the sequence identity of the respective genes. The investigation of natural populations of wild species such as S. vulgaris are necessary to reveal important aspects of CMS missed in domesticated crops, the traditional focus of the CMS studies.

• MeSH
• cytochromy b genetika   metabolismus   MeSH
• editace RNA MeSH
• genom mitochondriální * MeSH
• haplotypy MeSH
• homologní rekombinace * MeSH
• membránové glykoproteiny genetika   MeSH
• mitochondriální protonové ATPasy genetika   MeSH
• mitochondrie genetika   MeSH
• neplodnost rostlin genetika   MeSH
• otevřené čtecí rámce genetika   MeSH
• rostlinné proteiny genetika   MeSH
• Silene genetika   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH

An extraordinary variation in mitochondrial DNA sequence exists in angiosperm Silene vulgaris. The atp1 gene is flanked by very variable regions, as deduced from four completely sequenced mitochondrial genomes of this species. This diversity contributed to a highly variable transcript profile of this gene observed across S. vulgaris populations. We examined the atp1 transcript in the KOV mitochondrial genome and found three 5' ends, created most likely by the combination of transcription initiation and RNA processing. Most atp1 transcripts terminated about 70 bp upstream of the translation stop codon, which was present in only 10 % of them. Controlled crosses between a KOV mother and a geographically distant pollen donor (Krasnoyarsk, Russia) showed that nuclear background also affected atp1 transcription. The distant pollen donor introduced the factor(s) preventing the formation of a long 2,100 nt-transcript, because this long atp1 transcript reappeared in the progeny from self-crosses. The highly rearranged mitochondrial genomes with a variation in gene flanking regions make S. vulgaris an excellent model for the study of mitochondrial gene expression in plants.

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• buněčné jádro genetika   MeSH
• genetická transkripce * MeSH
• genom mitochondriální genetika   MeSH
• genom rostlinný genetika   MeSH
• genová přestavba genetika   MeSH
• křížení genetické MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• northern blotting MeSH
• otevřené čtecí rámce genetika   MeSH
• pyl genetika   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Silene genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oligomer aggregation of green-to-red photoconvertible fluorescent protein Eos (EosFP) is a natural feature of the wild‑type variant. The aim of the present study was to follow up mitochondrial nucleoid behavior under natural conditions of living cells transfected with mitochondrial single‑strand DNA‑binding protein (mtSSB) conjugated with EosFP. HEPG2 and SH‑SY5Y cells were subjected to lentiviral transfection and subsequently immunostained with anti‑DNA, anti‑transcription factor A, mitochondrial (TFAM) or anti‑translocase of the inner membrane 23 antibodies. Fluorescent microscopy, conventional confocal microscopy, superresolution biplane fluorescence photo-activation localization microscopy and direct stochastic optical reconstruction microscopy were used for imaging. In the two cell types, apparent couples of equally‑sized mtSSB‑EosFP‑visualized dots were observed. During the time course of the ongoing transfection procedure, however, a small limited number of large aggregates of mtSSB‑EosFP‑tagged protein started to form in the cells, which exhibited a great co‑localization with the noted coupled positions. Antibody staining and 3D immunocytochemistry confirmed that nucleoid components such as TFAM and DNA were co‑localized with these aggregates. Furthermore, the observed reduction of the mtDNA copy number in mtSSB‑EosFP‑transfected cells suggested a possible impairment of nucleoid function. In conclusion, the present study demonstrated that coupled nucleoids are synchronized by mtSSB‑EosFP overexpression and visualized through their equal binding capacity to mtSSB‑EosFP‑tagged protein. This observation suggested parallel replication and transcription activity of nucleoid couples native from a parental one. Preserved coupling in late stages of artificial EosFP‑mediated aggregation of tagged proteins suggested a rational manner of mitochondrial branching that may be cell-type specifically dependent on hierarchical nucleoid replication.

• MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• genetická transkripce MeSH
• genová dávka MeSH
• imunohistochemie MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• multimerizace proteinu * MeSH
• nádorové buněčné linie MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.

• MeSH
• buňky Hep G2 MeSH
• DNA vazebné proteiny metabolismus   MeSH
• doxorubicin MeSH
• ethidium MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• jaterní mitochondrie metabolismus   MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• poškození DNA MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• kyselina jantarová metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• proteasa La genetika   metabolismus   MeSH
• proteomika * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Roztroušená skleróza mozkomíšní (RS) je autoimunitní, imunopatologické a neurodegenerativní onemocnění centrální nervové soustavy. Nové možnosti, jak léčebně ovlivnit průběh RS, vycházejí z nových poznatků, které významně mění pohled na patogenezi RS. Jedná se především o úlohu mitochondriálních patologií a oxidačního stresu jako příčiny zánětlivé reakce, která je prostřednictvím inflamasomu indukována prozánětlivými cytokiny IL-1ß a IL-18. Významnou protektivní úlohu vůči negativním důsledkům oxidačního stresu sehrává transkripční faktor Nrf-2, který zesiluje transkripci stovek genů s antioxidačními, protizánětlivými a cytoprotektivními účinky. Dimetylfumaráty zprostředkují svůj léčebný účinek u nemocných s RS právě stimulací Nrf-2. Autoreaktivní T-lymfocyty migrují ze sekundárních lymfatických orgánů, které jsou místem indukce imunopatologie, do CNS. Migrace je usměrněna chemokiny a jejich receptory v gradientu sfingosinfosfátu. Expozice léčivu fingolimodu vede k internalizaci receptorů pro sfingosinfosfát a následnému omezení výstupu lymfoidních buněk ze sekundárních lymfatických orgánů. Výsledkem je významné omezení poškozujícího zánětu u nemocných s RS.

Multiple sclerosis (MS) is autoimmune, immunopathological, and neurodegenerative disease of the central nervous system. The substantial progress in our understanding of MS pathogenesis is reflected in the new means of treatment. The role of mitochondrial pathology and oxidative stress as a cause of inflammatory response is fully recognized now. In the presence of oxidative stress, there is the assembly of inflammasome with subsequent production of pluripotent proinflammatory cytokines IL-1ß and IL-18. The very substantial role in the control of oxidative stress is deserved to Nrf-2 transcription factor which in the presence of oxidative stress is regulating transcription of several hundred genes with antioxidative, antiinflammatory and cytoprotective activities. Dimethylfumarate, which is a newly introduced drug to treat MS, is targeting Nrf-2 transcription factor. Autoreactive T-cells which are primed and activated in secondary lymphoid organs have to migrate into the brain to demonstrate their immunopathological activities. The egress of lymphoid cells from secondary lymphoid organs is regulated by chemokines and gradient of sphingosine phosphate. The exposure of lymphoid cells to fingolimode which is now approved as effective in treatment of MS is modulating the sphingosine phosphate receptors on immune cells thus inhibiting migration of autoreactive T-cells outside of lymphoid organs.

• MeSH
• chemokiny imunologie   metabolismus   účinky léků   MeSH
• fumaráty farmakologie   terapeutické užití   MeSH
• lidé MeSH
• mitochondrie enzymologie   účinky léků   MeSH
• oxidační stres * imunologie   účinky léků   MeSH
• propylenglykoly terapeutické užití   MeSH
• receptory rozpoznávající vzory imunologie   účinky léků   MeSH
• roztroušená skleróza * farmakoterapie   imunologie   patofyziologie   MeSH
• sfingosin analogy a deriváty   imunologie   terapeutické užití   MeSH
• transkripční faktor NF-E2 farmakokinetika   farmakologie   terapeutické užití   MeSH
• zánět * imunologie   metabolismus   patofyziologie   MeSH
• Check Tag
• lidé MeSH

The present study was designed to characterize bovine oocytes with different meiotic competence and atresia levels in terms of their mitochondrial status. Oocyte subpopulations were recovered either from medium (MF) or small (SF) follicles and categorized as healthy, light-atretic and mid-atretic according to oocyte morphology. Mitochondrial activity, morphology and distribution, adenosine triphosphate (ATP) content and expression of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1) were assessed before (GV) and after (MII) maturation. The data were related to follicular size regardless of or with regard to oocyte atresia. Regardless of atresia, the MF subpopulation showed a significantly higher mitochondrial activity and frequency of oocytes with granulated mitochondria at GV and clustered mitochondria at MII than the SF subpopulation. With regard to atresia, mitochondrial activity decreased from healthy to mid-atretic oocytes in both MF and SF subpopulations at GV, but in the SF subpopulation at MII, the mitochondrial activity and frequency of oocytes with clustered mitochondria were significantly higher in light-atretic than in healthy oocytes. The light-atretic oocytes also produced more ATP than healthy ones in both SF and MF subpopulations. However, a significantly higher relative abundance of mRNA TFAM was found in SF than MF subpopulations at GV, and this difference remained in mid-atretic oocytes at MII. It can be concluded that meiotic competence and atresia level influence mitochondrial status of immature bovine oocytes. After maturation, healthy oocytes from medium follicles and light-atretic oocytes from small follicles were more developed in terms of mitochondrial status than the other oocytes.

• MeSH
• adenosintrifosfát analýza   MeSH
• DNA vazebné proteiny analýza   genetika   MeSH
• folikulární atrézie fyziologie   MeSH
• meióza * MeSH
• messenger RNA analýza   MeSH
• mitochondriální proteiny analýza   genetika   MeSH
• mitochondrie fyziologie   ultrastruktura   MeSH
• oocyty metabolismus   ultrastruktura   MeSH
• ovariální folikul chemie   MeSH
• skot * MeSH
• transkripční faktor NRF1 analýza   genetika   MeSH
• transkripční faktory analýza   genetika   MeSH
• zvířata MeSH
• Check Tag
• skot * MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial nucleoids are confined sites of mitochondrial DNA existing in complex clusters with the DNA-compacting mitochondrial (mt) transcription factor A (TFAM) and other accessory proteins and gene expression machinery proteins, such as a mt single-stranded-DNA-binding protein (mtSSB). To visualize nucleoid distribution within the mt reticular network, we have employed three-dimensional (3D) double-color 4Pi microscopy. The mt network was visualized in hepatocellular carcinoma HepG2 cells via mt-matrix-addressed GFP, while 3D immunocytochemistry of mtSSB was performed. Optimization of iso-surface computation threshold for nucleoid 4Pi images to 30 led to an average nucleoid diameter of 219 ± 110 and 224 ± 100 nm in glucose- and galactose-cultivated HepG2 cells (the latter with obligatory oxidative phosphorylation). We have positioned mtDNA nucleoids within the mt reticulum network and refined our model for nucleoid redistribution within the fragmented network--clustering of up to ten nucleoids in 2 μm diameter mitochondrial spheroids of a fragmented mt network, arising from an original 10 μm mt tubule of a 400 nm diameter. However, the theoretically fragmented bulk parts were observed most frequently as being reintegrated into the continuous mt network in 4Pi images. Since the predicted nucleoid counts within the bulk parts corresponded to the model, we conclude that fragmentation/reintegration cycles are not accompanied by mtDNA degradation or that mtDNA degradation is equally balanced by mtDNA replication.

• MeSH
• buněčné kultury MeSH
• buňky Hep G2 MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• imunohistochemie MeSH
• konfokální mikroskopie metody   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mitochondriální DNA genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• molekulární modely * MeSH
• počítačové zpracování obrazu MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Uveal melanoma (UM) is the most common primary intraocular tumour in adults. The most accurate prognostic factor of UM is classification by gene expression profiling. Currently, the role of epigenetics is much less defined compared to genetic mechanisms. We recently showed a strong prognostic role of the expression levels of histone variant macroH2A1 in UM patients. Here, we assessed the mechanistic effects of macroH2A1 on UM progression.UM cell lines were stably knocked down (KD) for macroH2A1, and proliferation and colony formation capacity were evaluated. Mitochondrial function was assayed through qPCR and HPLC analyses. Correlation between mitochondrial gene expression and cancer aggressiveness was studied using a bioinformatics approach.MacroH2A1 loss significantly attenuated UM cells proliferation and aggressiveness. Furthermore, genes involved in oxidative phosphorylation displayed a decreased expression in KD cells. Consistently, macroH2A1 loss resulted also in a significant decrease of mitochondrial transcription factor A (TFAM) expression, suggesting impaired mitochondrial replication. Bioinformatics analyses uncovered that the expression of genes involved in mitochondrial metabolism correlates with macroH2A1 and with cancer aggressiveness in UM patients. Altogether, our results suggest that macroH2A1 controls UM cells progression and it may represent a molecular target to develop new pharmacological strategies for UM treatment.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• histony nedostatek   MeSH
• lidé MeSH
• melanocyty metabolismus   MeSH
• melanom genetika   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky metabolismus   MeSH
• nádory uvey genetika   MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Placental oxidative stress has been implicated in pregnancy complications and previous work has shown that selenium can protect trophoblast mitochondria from oxidative stress. This report examines mitochondrial function and content in trophoblasts supplemented with selenium. METHODS: Swan-71, JEG-3 and BeWo cells and placental tissue were incubated with sodium selenite or selenomethionine. Mitochondrial function was examined in a respirometer. Mitochondrial content was determined using RT-PCR. The levels of the mitochondrial biogenesis markers selenoprotein H, PGC-1α and NRF-1 was examined by western blotting. RESULTS: Mitochondrial respiration was significantly enhanced post selenium supplementation in cells and tissues. Selenium supplementation increased mitochondrial content and up-regulated mitochondrial biogenesis mediators in cells. DISCUSSION: These results emphasise the importance of selenium in mitochondrial regeneration in trophoblasts.

• MeSH
• biogeneze organel * MeSH
• buněčné linie MeSH
• DNA vazebné proteiny metabolismus   MeSH
• faktor 1 související s NF-E2 metabolismus   MeSH
• lidé MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• placenta účinky léků   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• selen aplikace a dávkování   MeSH
• selenoproteiny metabolismus   MeSH
• těhotenství MeSH
• transkripční faktory metabolismus   MeSH
• trofoblasty účinky léků   metabolismus   MeSH
• upregulace účinky léků   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH