THE EFFECT OF ETHIDIUM BROMIDE ON MITOCHONDRIAL DNA SYNTHESIS AND MITOCHONDRIAL DNA STRUCTURE IN H<scp>E</scp>L<scp>A</scp> CELLS

Leibowitz, Robert D.

doi:10.1083/jcb.51.1.116

Cited by 69 publications

(35 citation statements)

References 10 publications

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“…Therefore, we also depleted mitochondrial DNA from WT macrophages using ethidium bromide. In agreement with previous reports (39,40), ethidium bromide treatment inhibited mitochondrial (Cox2) but not nuclear encoded DNA (Cs, Bhad) (Supplemental Figure 4B), reducing the expression of complex I, complex II, and complex IV by approximately 40% (Supplemental Figure 4C). Consistent with findings from β1 -/-macrophages, we found that lower levels of mitochondria led to reductions in fatty acid oxidation ( Figure 3C) and dramatically increased the phosphorylation of JNK ( Figure 3D).…”

Section: Figuresupporting

confidence: 80%

Hematopoietic AMPK β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity

Galić¹,

Fullerton²,

Schertzer³

et al. 2011

J. Clin. Invest.

308

320

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Individuals who are obese are frequently insulin resistant, putting them at increased risk of developing type 2 diabetes and its associated adverse health conditions. The accumulation in adipose tissue of macrophages in an inflammatory state is a hallmark of obesity-induced insulin resistance. Here, we reveal a role for AMPK β1 in protecting macrophages from inflammation under high lipid exposure. Genetic deletion of the AMPK β1 subunit in mice (referred to herein as β1 -/-mice) reduced macrophage AMPK activity, acetyl-CoA carboxylase phosphorylation, and mitochondrial content, resulting in reduced rates of fatty acid oxidation. β1 -/-macrophages displayed increased levels of diacylglycerol and markers of inflammation, effects that were reproduced in WT macrophages by inhibiting fatty acid oxidation and, conversely, prevented by pharmacological activation of AMPK β1-containing complexes. The effect of AMPK β1 loss in macrophages was tested in vivo by transplantation of bone marrow from WT or β1 -/-mice into WT recipients. When challenged with a high-fat diet, mice that received β1 -/-bone marrow displayed enhanced adipose tissue macrophage inflammation and liver insulin resistance compared with animals that received WT bone marrow. Thus, activation of AMPK β1 and increasing fatty acid oxidation in macrophages may represent a new therapeutic approach for the treatment of insulin resistance.

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Section: Figuresupporting

confidence: 80%

Hematopoietic AMPK β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity

Galić¹,

Fullerton²,

Schertzer³

et al. 2011

J. Clin. Invest.

308

320

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“…The column was washed with the same buffer until no more protein was eluted and then eluted with 5 column volumes of 25% formamide in 10 mM Tris-hydrochloride, pH 7.4, followed by 5 with an equal volume of complete Freund adjuvant, and 0.5 ml corresponding to material from 20 10-cm tissue culture dishes was injected intraperitoneally per BALB/c mouse. A similar booster injection in incomplete Freund adjuvant was given on day 14, and the mice were hyperimmunized by an intravenous injection of the same antigen in PBS on day 28. Mice were sacrificed 3 or 4 days later, the spleens were removed, and the lymphocytes were fused with SP2/0 myeloma cells.…”

Section: Methodsmentioning

confidence: 99%

“…Cultures were supplemented with penicillin-streptomycin, and used at subconfluent densities. Cells were labeled for 4 h with [35S]methionine at 10 ,uCi/ml in methionine-free medium containing 2% undialyzed fetal calf serum in the presence of actinomycin D (0.04 ,ug/ml) and ethidium bromide (1.5 ,ug/ml) added 30 min before addition of the label to suppress ribosomal (9,40,54) and mitochondrial (28,58) RNA and protein synthesis, respectively. Cell culture materials were from GIBCO Laboratories, and radiochemical reagents were obtained from New England Nuclear Corp.…”

Section: Methodsmentioning

confidence: 99%

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

Dreyfuss¹,

Adam²,

Choi³

1984

Mol. Cell. Biol.

336

168

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Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-p-Dribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.The complexes of proteins with heterogeneous nuclear RNA (hnRNA) (heterogeneous nuclear ribonucleoproteins [hnRNPs]) and with mRNA (messenger ribonucleoproteins [mRNPs]) are considered to be the functional assemblies which are involved in the synthesis and function of these polynucleotides in the cell (for a review, see reference 33). The proteins which form these complexes are, therefore, of key interest and are the focus of much research. The major difficulties in identifying RNPs by conventional isolation techniques stem from the fact that the criteria of copurification of certain proteins with polynucleotides are, in general, not sufficiently stringent to ascertain whether these are genuine RNP components in vivo. This is due to the fact that nonspecific interactions between RNA and proteins are likely to occur in vitro (2, 14, 38). UV cross-linking of RNA to protein in intact cells overcomes these difficulties and allows the identification of proteins which are in direct contact with hnRNA and mRNA in vivo (10,34,35,50,51,53).The in vivo UV cross-linking approach relies on the fact that UV light of sufficient intensity generates photoreactive species of RNA which react virtually indiscriminately with molecu...

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“…Mitochondria and mtDNA are believed to be closely associated with cell growth, because mitochondria are organelles that produce chemical energy in the form of ATP (Kotsifas et al, 2003;Zhu et al, 1997). Ethidium bromide (EtBr) is known to be a potent inhibitor of mtDNA replication and transcription in mammalian cultured cells (Nass, 1970;Leibowitz, 1971;Zylber et al, 1969) and avian cultured cells (Desjardins et al, 1985;Desjardins et al, 1989), but it does not substantially affect the synthesis of nuclear DNA (Radask et al, 1971;Nass, 1972;LeblondLarouche et al, 1979). In cultures of Dictyostelium Ax-3 cells, EtBr actually inhibits any new synthesis of mtDNA (Firtel and Bonner, 1972;Kobilinsky and Beattie, 1977), and growth of Ax-3 cells stops after one or two generations in the presence of 10 µg/ml EtBr (Stuchell et al, 1973).…”

mentioning

confidence: 99%

The necessity of mitochondrial genome DNA for normal development ofDictyosteliumcells

Chida

Yamaguchi

Amagai

et al. 2004

Journal of Cell Science

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Most unexpectedly, there is now increasing evidence that mitochondria have novel and crucial functions in the regulatory machinery of the growth/differentiation transition, cell-type determination, cellular movement and pattern formation. Here we created ρΔ cells with a reduced amount (about 1/4) of mitochondrial DNA (mtDNA) from Dictyostelium discoideum Ax-2 cells, by exposing Ax-2 cells to ca. 30 μg/ml of ethidium bromide (EtBr) in axenic growth medium. Importantly, the ρΔ cells exhibited a series of fascinating behaviors: when they were starved, they showed a marked delay of differentiation and stopped their development at the slug stage, thus failing to construct fruiting bodies. Moreover, cell patterning and cell-type proportioning were found to be greatly modified in slugs (referred to as ρΔ slugs) derived from ρΔ cells. That is, prestalk differentiation was significantly enhanced in ρΔ slugs, while prespore differentiation was markedly inhibited. In addition, the clear anterior prestalk/posterior prespore pattern was considerably disturbed in ρΔ slugs, presumably because of incomplete sorting between the two types of differentiated cells. After the assay of phototaxis, ρΔ slugs also exhibited highly disordered movement towards the light source. Taken together, these results suggest that mtDNA might have important multiple functions in a variety of cellular processes during Dictyostelium development.

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THE EFFECT OF ETHIDIUM BROMIDE ON MITOCHONDRIAL DNA SYNTHESIS AND MITOCHONDRIAL DNA STRUCTURE IN HELA CELLS

Cited by 69 publications

References 10 publications

Hematopoietic AMPK β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity

Hematopoietic AMPK β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

The necessity of mitochondrial genome DNA for normal development ofDictyosteliumcells

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