scholarly journals Methods for simultaneous and quantitative isolation of mitochondrial DNA, nuclear DNA and RNA from mammalian cells

BioTechniques ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 436-442
Author(s):  
Jean Nakhle ◽  
Tülin Özkan ◽  
Kateřina Lněničková ◽  
Philippe Briolotti ◽  
Marie-Luce Vignais
Keyword(s):  
Stem Cells ◽  
Mitochondrial Dna ◽  
Cell Line ◽  
Mammalian Cells ◽  
Nuclear Dna ◽  
Housekeeping Genes ◽  
Cell Types ◽  
Hepg2 Cell Line ◽  
Primary Glioblastoma ◽  
Dna And Rna

The aim of this study was to assess two protocols for their capacities to simultaneously isolate RNA, mtDNA and ncDNA from mammalian cells. We compared the Invitrogen TRIzol-based method and Qiagen DNeasy columns, using the HepG2 cell line and human primary glioblastoma stem cells. Both methods allowed the isolation of all three types of nucleic acids and provided similar yields in mtDNA. However, the yield in ncDNA was more than tenfold higher on columns, as observed for both cell types. Conversely, the TRIzol method proved more reproducible and was the method of choice for isolating RNA from glioblastoma cells, as demonstrated for the housekeeping genes RPLP0 and RPS9.

scholarly journals Mitochondrial DNA Heteroplasmy as an Informational Reservoir Dynamically Linked to Metabolic and Immunological Processes Associated with COVID-19 Neurological Disorders

2021 ◽  
Author(s):  
George B. Stefano ◽  
Richard M. Kream
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Function ◽  
Neurological Disorders ◽  
Nuclear Dna ◽  
Mitochondrial Dynamics ◽  
Cell Types ◽  
Tissue Specific ◽  
Copy Numbers ◽  
The Central Nervous System ◽  
Mitochondrial Dna Heteroplasmy

AbstractMitochondrial DNA (mtDNA) heteroplasmy is the dynamically determined co-expression of wild type (WT) inherited polymorphisms and collective time-dependent somatic mutations within individual mtDNA genomes. The temporal expression and distribution of cell-specific and tissue-specific mtDNA heteroplasmy in healthy individuals may be functionally associated with intracellular mitochondrial signaling pathways and nuclear DNA gene expression. The maintenance of endogenously regulated tissue-specific copy numbers of heteroplasmic mtDNA may represent a sensitive biomarker of homeostasis of mitochondrial dynamics, metabolic integrity, and immune competence. Myeloid cells, monocytes, macrophages, and antigen-presenting dendritic cells undergo programmed changes in mitochondrial metabolism according to innate and adaptive immunological processes. In the central nervous system (CNS), the polarization of activated microglial cells is dependent on strategically programmed changes in mitochondrial function. Therefore, variations in heteroplasmic mtDNA copy numbers may have functional consequences in metabolically competent mitochondria in innate and adaptive immune processes involving the CNS. Recently, altered mitochondrial function has been demonstrated in the progression of coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Accordingly, our review is organized to present convergent lines of empirical evidence that potentially link expression of mtDNA heteroplasmy by functionally interactive CNS cell types to the extent and severity of acute and chronic post-COVID-19 neurological disorders.

scholarly journals Functions of p53 in pluripotent stem cells

2019 ◽  
Vol 11 (1) ◽  
pp. 71-78 ◽  
Author(s):  
Xuemei Fu ◽  
Shouhai Wu ◽  
Bo Li ◽  
Yang Xu ◽  
Jingfeng Liu
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Mammalian Cells ◽  
Genomic Stability ◽  
Cell Types ◽  
The Body ◽  
Great Promise ◽  
Primary Role ◽  
Cycle Arrest ◽  
Clinical Potential

Abstract Pluripotent stem cells (PSCs) are capable of unlimited self-renewal in culture and differentiation into all functional cell types in the body, and thus hold great promise for regenerative medicine. To achieve their clinical potential, it is critical for PSCs to maintain genomic stability during the extended proliferation. The critical tumor suppressor p53 is required to maintain genomic stability of mammalian cells. In response to DNA damage or oncogenic stress, p53 plays multiple roles in maintaining genomic stability of somatic cells by inducing cell cycle arrest, apoptosis, and senescence to prevent the passage of genetic mutations to the daughter cells. p53 is also required to maintain the genomic stability of PSCs. However, in response to the genotoxic stresses, a primary role of p53 in PSCs is to induce the differentiation of PSCs and inhibit pluripotency, providing mechanisms to maintain the genomic stability of the self-renewing PSCs. In addition, the roles of p53 in cellular metabolism might also contribute to genomic stability of PSCs by limiting oxidative stress. In summary, the elucidation of the roles of p53 in PSCs will be a prerequisite for developing safe PSC-based cell therapy.

scholarly journals Efficient selection and characterization of mutants of a human cell line which are defective in mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase.

1995 ◽  
Vol 15 (2) ◽  
pp. 964-974 ◽  
Author(s):  
G Hofhaus ◽  
G Attardi
Keyword(s):  
Mitochondrial Dna ◽  
Cell Line ◽  
Human Cell ◽  
Complex I ◽  
Mammalian Cells ◽  
Nadh Dehydrogenase ◽  
Frameshift Mutation ◽  
Human Cell Line ◽  
Mtdna Mutation ◽  
Selection Scheme

The mitochondrial NADH dehydrogenase (complex I) in mammalian cells is a multimeric enzyme consisting of approximately 40 subunits, 7 of which are encoded in mitochondrial DNA (mtDNA). Very little is known about the function of these mtDNA-encoded subunits. In this paper, we describe the efficient isolation from a human cell line of mutants affected in any of these subunits. In the course of analysis of eight mutants of the human cell line VA2B selected for their resistance to high concentrations of the complex I inhibitor rotenone, seven were found to be respiration deficient, and among these, six exhibited a specific defect of complex I. Transfer of mitochondria from these six mutants into human mtDNA-less cells revealed, surprisingly, in all cases a cotransfer of the complex I defect but not of the rotenone resistance. This result indicated that the rotenone resistance resulted from a nuclear mutation, while the respiration defect was produced by an mtDNA mutation. A detailed molecular analysis of the six complex I-deficient mutants revealed that two of them exhibited a frameshift mutation in the ND4 gene, in homoplasmic or in heteroplasmic form, resulting in the complete or partial loss, respectively, of the ND4 subunit; two other mutants exhibited a frameshift mutation in the ND5 gene, in near-homoplasmic or heteroplasmic form, resulting in the ND5 subunit being undetectable or strongly decreased, respectively. It was previously reported (G. Hofhaus and G. Attardi, EMBO J. 12:3043-3048, 1993) that the mutant completely lacking the ND4 subunit exhibited a total loss of NADH:Q1 oxidoreductase activity and a lack of assembly of the mtDNA-encoded subunits of complex I. By contrast, in the mutant characterized in this study in which the ND5 subunit was not detectable and which was nearly totally deficient in complex I activity, the capacity to assemble the mtDNA-encoded subunits of the enzyme was preserved, although with a decreased efficiency or a reduced stability of the assembled complex. The two remaining complex I-deficient mutants exhibited a normal rate of synthesis and assembly of the mtDNA-encoded subunits of the enzyme, and the mtDNA mutation(s) responsible for their NADH dehydrogenase defect remains to be identified. The selection scheme used in this work has proven to be very valuable for the isolation of mutants from the VA2B cell line which are affected in different mtDNA-encoded subunits of complex I and may be applicable to other cell lines.

scholarly journals Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

2009 ◽  
Vol 53 (7) ◽  
pp. 3056-3064 ◽  
Author(s):  
Walter Rangel Lopes de Campos ◽  
Dayaneethie Coopusamy ◽  
Lynn Morris ◽  
Bongani M. Mayosi ◽  
Makobetsa Khati
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mitochondrial Dna ◽  
Reverse Transcriptase ◽  
Mononuclear Cells ◽  
Nuclear Dna ◽  
Cell Types ◽  
Reverse Transcriptase Inhibitors ◽  
Human Cardiomyocytes ◽  
Immunodeficiency Virus

ABSTRACT The long-term cumulative cytotoxicity of antiretrovirals (ARVs) is among the major causes of treatment failure in patients infected with human immunodeficiency virus (HIV) and patients with AIDS. This calls for the development of novel ARVs with less or no cytotoxicity. In the present study, we compared the cytotoxic effects of a cross-clade HIV type 1-neutralizing aptamer called B40 with those of a panel of nonnucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), and the entry inhibitor (EI) T20 in human cardiomyocytes and peripheral blood mononuclear cells. An initial screen in which cell death was used as the end-point measurement revealed that the B40 aptamer and T20 were the only test molecules that had insignificant (0.61 < P < 0.92) effects on the viability of both cell types at the maximum concentration used. PIs were the most toxic class (0.001 < P < 0.00001), followed by NNRTIs and NRTIs (0.1 < P < 0.00001). Further studies revealed that B40 and T20 did not interfere with the cellular activity of the cytochrome P450 3A4 enzyme (0.78 < P < 0.24) or monoamine oxidases A and B (0.83 < P < 0.56) when the activities of the enzymes were compared to those in untreated controls of both cell types. Mitochondrion-initiated cellular toxicity is closely associated with the use of ARVs. Therefore, we used real-time PCR to quantify the relative ratio of mitochondrial DNA to nuclear DNA as a marker of toxicity. The levels of mitochondrial DNA remained unchanged in cells exposed to the B40 aptamer compared to the levels in untreated control cells (0.5 > P > 0.06). These data support the development of B40 and related EI aptamers as new ARVs with no cytotoxicity at the estimated potential therapeutic dose.

scholarly journals Sequence analysis of mitochondrial DNA in a mouse cell line resistant to chloramphenicol and oligomycin.

1983 ◽  
Vol 3 (10) ◽  
pp. 1694-1702 ◽  
Author(s):  
E F Slott ◽  
R O Shade ◽  
R A Lansman
Keyword(s):  
Mitochondrial Dna ◽  
Sequence Analysis ◽  
Cell Line ◽  
Mammalian Cells ◽  
Mouse Cell ◽  
Mitochondrial Atpase ◽  
Dna Molecules ◽  
Single Nucleotide Change ◽  
In The Wild ◽  
Atpase 6

A mouse L-cell line, designated 111-OB3, is described which is resistant to two drugs, chloramphenicol and oligomycin. The cells contain two types of mitochondrial DNA molecules, in roughly equal proportions, which differ in that one is cleaved by endonuclease EcoRI at a novel site within the coding sequence for subunit 6 of the mitochondrial ATPase (ATPase-6). Sequence analysis reveals that the cleavage site was created by a single transversion which predicts a replacement of valine in the wild-type ATPase-6 by glutamic acid. The replacement occurs in a hydrophobic amino acid sequence which is highly conserved in mouse, human, and bovine proteins. The position of the replacement is similar to a substitution observed in one class of yeast mutants resistant to oligomycin. Both of the mitochondrial DNA molecules in 111-OB3 also have a single nucleotide change in the gene encoding the large (16S) rRNA. These observations are consistent with the hypothesis that oligomycin resistance in mammalian cells can be cytoplasmically determined and can result from alterations in ATPase-6. The appearance of the mutation before selection in oligomycin suggests a model for the origin of mitochondrial mutations in mammalian cells.

scholarly journals MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jeroen Witteveldt ◽  
Lisanne I Knol ◽  
Sara Macias
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Mammalian Cells ◽  
Viral Infections ◽  
Embryonic Stem ◽  
Cell Types ◽  
Interferon Response ◽  
Central Component ◽  
Type I ◽  
Antiviral Mechanism

When mammalian cells detect a viral infection, they initiate a type I interferon (IFNs) response as part of their innate immune system. This antiviral mechanism is conserved in virtually all cell types, except for embryonic stem cells (ESCs) and oocytes which are intrinsically incapable of producing IFNs. Despite the importance of the IFN response to fight viral infections, the mechanisms regulating this pathway during pluripotency are still unknown. Here we show that, in the absence of miRNAs, ESCs acquire an active IFN response. Proteomic analysis identified MAVS, a central component of the IFN pathway, to be actively silenced by miRNAs and responsible for suppressing IFN expression in ESCs. Furthermore, we show that knocking out a single miRNA, miR-673, restores the antiviral response in ESCs through MAVS regulation. Our findings suggest that the interaction between miR-673 and MAVS acts as a switch to suppress the antiviral IFN during pluripotency and present genetic approaches to enhance their antiviral immunity.

scholarly journals Heteroplasmy concordance between mitochondrial DNA and RNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ruoyu Zhang ◽  
Kiichi Nakahira ◽  
Augustine M. K. Choi ◽  
Zhenglong Gu
Keyword(s):  
Mitochondrial Dna ◽  
Cell Line ◽  
Mitochondrial Rna ◽  
Sequencing Data ◽  
Cell Functions ◽  
Dna And Rna ◽  
Public Data ◽  
Rapid Changes ◽  
Sequencing Studies

Abstract Mitochondrial DNA (mtDNA) heteroplasmies are associated with various diseases but the transmission of heteroplasmy from mtDNA to mitochondrial RNA (mtRNA) remains unclear. We compared heteroplasmies in mtRNA from 446 human B-lymphoblastoid cell lines to their corresponding mtDNA using deep sequencing data from two independent studies. We observed 2786 heteroplasmies presenting in both DNA and RNA at 1% frequency cutoff. Among them, the frequencies of 2427 (87.1%) heteroplasmies were highly consistent (less than 5% frequency difference) between DNA and RNA. To validate these frequency consistencies, we isolated DNA and RNA simultaneously from GM12282 cell line used in those two sequencing studies, and resequenced its heteroplasmy sites. Interestingly, we also observed the rapid changes of heteroplasmy frequencies during 4 weeks of the cell culture: the frequencies at Day 14 increased by >25% than those at Day 0. However, the heteroplasmy frequencies from the same time point were highly consistent. In summary, our analysis on public data together with in vitro study indicates that the heteroplasmies in DNA can be transcribed into RNA with high fidelity. Meanwhile, the observed rapid-changing heteroplasmy frequency can potentially disturb cell functions, which could be an overlooked confounding factor in cell line related studies.

scholarly journals A Sweet Potion to Put Embryonic Stem Cells to Sleep

2009 ◽  
Vol 9 ◽  
pp. 236-249 ◽  
Author(s):  
Kaushik D. Deb
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Intercellular Transport ◽  
Ambient Temperatures ◽  
Naturally Occurring ◽  
Therapeutic Properties ◽  
Temperature Storage

Human embryonic stem cells (hESCs) are rapidly revolutionizing the areas of drug screening and therapy. In view of their applications and high operational costs at global multicentric setups, the ability to store and transport hESCs and derivatives under ambient temperatures, and their cryopreservation without compromising the stemness, function, and viability, is becoming imperative. Here we discuss the need for a natural cryoprotectant and biopreservative with a potential to improve cryopreservation, ambient temperature storage, and shipping of hESCs and derivatives. Trehalose, a naturally occurring disaccharide with therapeutic properties, protects the integrity of cells against desiccation, dehydration, and extreme heat or cold, and has been successfully tested for some somatic stem cell types. However, the biggest setback is the inability of mammalian cells to internalize trehalose. Here we review the methods being developed at different laboratories to facilitate its intercellular transport and advocate the need for similar advances in hESCs.

Identification of toxic mineral dusts using mammalian cells

1980 ◽  
Vol 71 (3) ◽  
pp. 181-184 ◽  
Author(s):  
R. Davies ◽  
M. Chamberlain ◽  
R. C. Brown ◽  
D. M. Griffiths
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Alveolar Type ◽  
Mineral Dusts ◽  
Potential Pathogenicity

ABSTRACTCell culture systems have been developed to assess the potential pathogenicity of mineral dusts. The in vitro cytotoxicities of a variety of dusts towards mouse peritoneal macrophages, Chinese hamster lung cells (V79 cell line) and human alveolar type II cells (A549 cell line) were investigated.Non-pathogenic dusts were found to be inert in vitro. Fibrogenic non-fibrous dusts such as silica were only cytotoxic towards macrophages. Fibrous dusts which are both fibrogenic and carcinogenic in vivo are cytotoxic towards all three cell types, their cytotoxicity being dependent on fibre size. The size range important for the observed biological effect is longer than about 8 μm and thinner than about 1·5 μm.

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