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Investigative Ophthalmology & Visual... Nov 2023The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the...
PURPOSE
The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the proliferative response of mammalian Müller glia, we comparatively analyzed the expression and function of nestin, an intermediate filament protein established as a neural stem cell marker, in the mouse and rat retinas after injury.
METHODS
Nestin expression in the retinas of C57BL/6 mice and Wistar rats after methyl methanesulfonate (MMS)-induced photoreceptor injury was examined by immunofluorescence and Western blotting. Adeno-associated virus (AAV)-delivered control and nestin short hairpin RNA (shRNA) were intravitreally injected to rats and Müller glia proliferation after MMS-induced injury was analyzed by BrdU incorporation and immunofluorescence. Photoreceptor removal and microglia/macrophage infiltration were also analyzed by immunofluorescence.
RESULTS
Rat Müller glia re-entered the cell cycle and robustly upregulated nestin after injury whereas Müller glia proliferation and nestin upregulation were not observed in mice. In vivo knockdown of nestin in the rat retinas inhibited Müller glia proliferation while transiently stimulating microglia/macrophage infiltration and phagocytic removal of dead photoreceptors.
CONCLUSIONS
Our findings suggest a critical role for nestin in the regulation of Müller glia proliferation after retinal injury and highlight the importance of cross species analysis to identify the molecular mechanisms regulating the injury responses of the mammalian retina.
Topics: Animals; Mice; Rats; Cell Proliferation; Eye Injuries; Methyl Methanesulfonate; Mice, Inbred C57BL; Nestin; Rats, Wistar; Neuroglia
PubMed: 37934159
DOI: 10.1167/iovs.64.14.8 -
Bulletin of Experimental Biology and... Jan 2018The effect of usnic acid enantiomers on the genotoxic effects of dioxidine and methyl methanesulfonate was studied in vitro in human peripheral blood lymphocytes by the...
The effect of usnic acid enantiomers on the genotoxic effects of dioxidine and methyl methanesulfonate was studied in vitro in human peripheral blood lymphocytes by the DNA comet method. We found that usnic acid enantiomers in a concentration range of 0.01-1.00 μM demonstrated pronounced antigenotoxic activity and reduced DNA damage induced by genotoxicants by 37-70%. In the same concentration range, the test enantiomers reduced the level of atypical DNA comets (hedgehogs) induced by genotoxicants by 23-61%. The test compounds did not modulate the effects of genotoxicants in a concentration of 10 μM and potentiated them in a concentration of 100 μM. The modifying activity of usnic acid did not depend on spatial configuration and on the used model genotoxicant.
Topics: Benzofurans; Comet Assay; DNA Fragmentation; Hormesis; Humans; Leukocytes, Mononuclear; Lichens; Methyl Methanesulfonate; Mutagens; Protective Agents; Quinoxalines; Stereoisomerism
PubMed: 29308568
DOI: 10.1007/s10517-018-3979-z -
Nanomaterials (Basel, Switzerland) Dec 2022Research on nano- and micro-plastic particles (NMPPs) suggests their potential threat to human health. Some studies have even suggested genotoxic effects of NMPP...
Research on nano- and micro-plastic particles (NMPPs) suggests their potential threat to human health. Some studies have even suggested genotoxic effects of NMPP exposure, such as micronuclei (MN) formation, while others found the opposite. To clarify the ability of NMPP to induce MN formation, we used non-malignant HaCaT keratinocytes and exposed these to a variety of polystyrene (PS) and poly methyl methacrylate (PMMA) particle types at different concentrations and three different sizes. Investigations were performed following acute (one day) and chronic exposure (five weeks) against cytotoxic (amino-modified NMPPs) and genotoxic (methyl methanesulfonate, MMS) positive controls. An optimized high-content imaging workflow was established strictly according to OECD guidelines for analysis. Algorithm-based object segmentation and MN identification led to computer-driven, unsupervised quantitative image analysis results on MN frequencies among the different conditions and thousands of cells per condition. This could only be realized using accutase, allowing for partial cell detachment for optimal identification of bi-nucleated cells. Cytotoxic amino-modified particles were not genotoxic; MMS was both. During acute and long-term studies, PS and PMMA particles were neither toxic nor increased MN formation, except for 1000 nm PS particles at the highest concentration of unphysiological 100 µg/mL. Interestingly, ROS formation was significantly decreased in this condition. Hence, most non-charged polymer particles were neither toxic nor genotoxic, while aminated particles were toxic but not genotoxic. Altogether, we present an optimized quantitative imaging workflow applied to a timely research question in environmental toxicity.
PubMed: 36558315
DOI: 10.3390/nano12244463 -
Parasitology Oct 2018Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly...
Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly factors. We investigated putative participants in the biogenesis of the reduced organellar ribosomes of Plasmodium falciparum and identified homologues of two assembly GTPases - EngA and Obg that were found in mitochondria. Both are indispensable in bacteria and P. berghei EngA is among the 'essential' parasite blood stage proteins identified recently. PfEngA and PfObg1 interacted with parasite mitoribosomes in vivo. GTP stimulated PfEngA interaction with the 50S subunit of Escherichia coli surrogate ribosomes. Although PfObg1-ribosome interaction was independent of nucleotide binding, GTP hydrolysis by PfObg1 was enhanced upon ribosomal association. An additional function for PfObg1 in mitochondrial DNA transactions was suggested by its specific interaction with the parasite mitochondrial genome in vivo. Deletion analysis revealed that the positively-charged OBG (spoOB-associated GTP-binding protein) domain mediates DNA-binding. A role for PfEngA in mitochondrial genotoxic stress response was indicated by its over-expression upon methyl methanesulfonate-induced DNA damage. PfEngA had lower sensitivity to an E. coli EngA inhibitor suggesting differences with bacterial counterparts. Our results show the involvement of two important GTPases in P. falciparum mitochondrial function, with the first confirmed localization of an EngA homologue in eukaryotic mitochondria.
Topics: GTP Phosphohydrolases; Mitochondria; Plasmodium falciparum; Protein Transport; Protozoan Proteins; Ribosomes
PubMed: 29642957
DOI: 10.1017/S0031182018000501 -
Molecular Microbiology Oct 2023Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While...
Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While recent work has revealed that Pso2/Snm1 is dual-localized in the nucleus and mitochondria, it remains unclear whether cell-intrinsic and -extrinsic factors regulate its subcellular localization and function. Herein, we show that Pso2 undergoes ubiquitination and phosphorylation, but not SUMOylation, in unstressed cells. Unexpectedly, we found that methyl methanesulfonate (MMS), rather than ICL-forming agents, induced robust SUMOylation of Pso2 on two conserved residues, K97 and K575, and that SUMOylation markedly increased its abundance in the mitochondria. Reciprocally, SUMOylation had no discernible impact on Pso2 translocation to the nucleus, despite the presence of steady-state levels of SUMOylated Pso2 across the cell cycle. Furthermore, substitution of the invariant residues K97 and K575 by arginine in the Pso2 SUMO consensus motifs severely impaired SUMOylation and abolished its translocation to the mitochondria of MMS-treated wild type cells, but not in unstressed cells. We demonstrate that whilst Siz1 and Siz2 SUMO E3 ligases catalyze Pso2 SUMOylation, the former plays a dominant role. Notably, we found that the phenotypic characteristics of the SUMOylation-defective mutant Pso2 closely mirrored those observed in the Pso2Δ petite mutant. Additionally, leveraging next-generation sequencing analysis, we demonstrate that Pso2 mitigates MMS-induced damage to mitochondrial DNA (mtDNA). Viewed together, our work offers previously unknown insights into the link between genotoxic stress-induced SUMOylation of Pso2 and its preferential targeting to the mitochondria, as well as its role in attenuating MMS-induced mtDNA damage.
Topics: Humans; Saccharomyces cerevisiae; Methyl Methanesulfonate; DNA, Mitochondrial; Sumoylation; Saccharomyces cerevisiae Proteins; Endodeoxyribonucleases; DNA Damage; Mitochondria; Translocation, Genetic; Ubiquitin-Protein Ligases
PubMed: 37649278
DOI: 10.1111/mmi.15145 -
Talanta Jan 2021Alkylated DNA adducts are the most important and common form of DNA damage at the molecular level. In addition to known alkylated DNA adducts, many unknown DNA adducts...
Alkylated DNA adducts are the most important and common form of DNA damage at the molecular level. In addition to known alkylated DNA adducts, many unknown DNA adducts remain to be discovered. A prediction-driven MRM profiling MS strategy has been established for the rapid discovery of unknown DNA adducts induced by sulfonates. The innovative aspects and core of this strategy are the construction of the prediction MRM list, which includes 36 possible precursor ion and characteristic product ion transitions of DNA adducts based on MS fragmentation patterns, and then unknown DNA adducts 7-propyl guanine and 7-butyl guanine were discovered based on the diagnostic MRM signals of the DNA samples, and subsequently confirmed using high-resolution MS data and synthetic standards for the first time. Furthermore, DNA adducts, including newly found adducts in a human cell model and rat tissues after nitrosamine and sulfonate exposure, were unambiguously investigated by a UHPLC-MS/MS method. As a result, different alkyl methanesulfonates, including methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), PMS and BMS, all lead to the formation of 7MeG in addition to their own specific alkylation DNA adducts. The ester group of the sulfonate determines the specific types of DNA adducts produced, and the sulfonate might undergo transesterification with the methyl donors that commonly exist in eukaryotic organisms such as SAM, resulting in the formation of MMS, which induce the generation of methyl DNA adducts after EMS, PMS and BMS exposure. Furthermore, similar DNA adduct profiles were presented in both human cells and rat tissues. This approach could be useful in the future for probing unknown DNA adducts and simultaneously profiling both known and unknown DNA adducts in both in vitro to in vivo settings to evaluate potential genotoxicities and cancer risks to populations exposed to genotoxins.
Topics: Alkylation; Animals; DNA Adducts; Methyl Methanesulfonate; Mutagens; Rats; Tandem Mass Spectrometry
PubMed: 33167213
DOI: 10.1016/j.talanta.2020.121500 -
Plant Cell Reports Aug 2023SMC5/6 complex subunit OsMMS21 is involved in cell cycle and hormone signaling and required for stem cell proliferation during shoot and root development in rice. The...
SMC5/6 complex subunit OsMMS21 is involved in cell cycle and hormone signaling and required for stem cell proliferation during shoot and root development in rice. The structural maintenance of chromosome (SMC)5/6 complex is required for nucleolar integrity and DNA metabolism. Moreover, METHYL METHANESULFONATE SENSITIVITY GENE 21 (MMS21), a SUMO E3 ligase that is part of the SMC5/6 complex, is essential for the root stem cell niche and cell cycle transition in Arabidopsis. However, its specific role in rice remains unclear. Here, OsSMC5 and OsSMC6 single heterozygous mutants were generated using CRISPR/Cas9 technology to elucidate the function of SMC5/6 subunits, including OsSMC5, OsSMC6, and OsMMS21, in cell proliferation in rice. ossmc5/ + and ossmc6/ + heterozygous single mutants did not yield homozygous mutants in their progeny, indicating that OsSMC5 and OsSMC6 both play necessary roles during embryo formation. Loss of OsMMS21 caused severe defects in both the shoot and roots in rice. Transcriptome analysis showed a significant decrease in the expression of genes involved in auxin signaling in the roots of osmms21 mutants. Moreover, the expression levels of the cycB2-1 and MCM genes, which are involved the cell cycle, were significantly lower in the shoots of the mutants, indicating that OsMMS21 was involved in both hormone signaling pathways and the cell cycle. Overall, these findings indicate that the SUMO E3 ligase OsMMS21 is required for both shoot and root stem cell niches, improving the understanding of the function of the SMC5/6 complex in rice.
Topics: Saccharomyces cerevisiae Proteins; Oryza; Cell Cycle Proteins; SUMO-1 Protein; Ubiquitin-Protein Ligases; Cell Division; Hormones
PubMed: 37178216
DOI: 10.1007/s00299-023-03030-9 -
Life Science Alliance Sep 2021DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of is lethal, and...
DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of is lethal, and compromising the Pol3-Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying Δ. We have identified alanine-substitutions in that suppress these deficiencies in Δ cells. We characterize two mutants, and , which map to a solvent-exposed loop that mediates Pol31-Pol3 and Pol31-Rev3 interactions. The substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as , leads to reduced Pol3 and Pol31 protein levels, which are restored by -W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in Δ cells is not restored by , highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that Δ and indirectly destabilize DNA pol α and pol ε at stalled forks.
Topics: Binding Sites; DNA Polymerase III; DNA Repair; DNA Replication; Fungal Proteins; Multiprotein Complexes; Protein Binding; Protein Subunits; Yeasts
PubMed: 34226278
DOI: 10.26508/lsa.202101138 -
Mutation Research. Genetic Toxicology... Sep 2019In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of... (Comparative Study)
Comparative Study
In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of human toxicity of industrial chemicals and environmental pollutants. In this study we compared cytotoxic and genotoxic responses induced by chemicals in 2D and 3D spheroidal cultures of the human liver cancer cell line HepG2. HepG2 spheroids were prepared by hanging drop technology. Both 3D spheroids and 2D monolayer cultures were exposed to different chemicals (colchicine, chlorpromazine hydrochloride or methyl methanesulfonate) for geno- and cytotoxicity studies. Cytotoxicity was investigated by alamarBlue assay, flow cytometry and confocal imaging. DNA damage was investigated by the comet assay with and without Fpg enzyme for detection of DNA strand breaks and oxidized or alkylated base lesions. The results from the cyto- and genotoxicity tests showed differences in sensitivity comparing the 2D and 3D HepG2 models. This study shows that human 3D spheroidal hepatocellular cultures can be successfully applied for genotoxicity testing by the comet assay and represent a promising advanced in vitro model for toxicity testing.
Topics: Cell Culture Techniques; Cell Survival; Chlorpromazine; Colchicine; Comet Assay; DNA Damage; DNA, Neoplasm; Guanine; Hep G2 Cells; Humans; Hydrogen Peroxide; Laboratory Proficiency Testing; Methyl Methanesulfonate; Sensitivity and Specificity; Spheroids, Cellular
PubMed: 31561895
DOI: 10.1016/j.mrgentox.2019.03.006 -
Mutation Research. Genetic Toxicology... Apr 2023The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis....
The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.
Topics: Mice; Animals; Cisplatin; Mice, Knockout; Cell Division; DNA Damage; Carcinogenesis; Alpha-Ketoglutarate-Dependent Dioxygenase FTO
PubMed: 37003652
DOI: 10.1016/j.mrgentox.2023.503608