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Cell Death & Disease Jul 2023Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining...
Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining hypoxia-induced mitochondrial protein aggregation in C. elegans, we found that sublethal hypoxia, sodium azide, or heat shock-induced abundant ethidium bromide staining mitochondrial granules that preceded evidence of protein aggregation. Genetic manipulations that reduce cellular and organismal hypoxic death block the formation of these mitochondrial stress granules (mitoSG). Knockdown of mitochondrial nucleoid proteins also blocked the formation of mitoSG by a mechanism distinct from the mitochondrial unfolded protein response. Lack of the major mitochondrial matrix protease LONP-1 resulted in the constitutive formation of mitoSG without external stress. Ethidium bromide-staining RNA-containing mitochondrial granules were also observed in rat cardiomyocytes treated with sodium azide, a hypoxia mimetic. Mitochondrial stress granules are an early mitochondrial pathology controlled by LONP and the nucleoid, preceding hypoxia-induced protein aggregation.
Topics: Animals; Rats; Caenorhabditis elegans; Protein Aggregates; Ethidium; Sodium Azide; Stress Granules; Hypoxia; Mitochondrial Proteins
PubMed: 37468471
DOI: 10.1038/s41419-023-05988-6 -
Journal of Neuroinflammation Oct 2022Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of...
BACKGROUND
Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain.
METHODS
A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake.
RESULTS
CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression.
CONCLUSION
In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.
Topics: Adenosine Triphosphate; Animals; Carbenoxolone; Connexins; Cytokines; Ethidium; Hyperalgesia; Lipopolysaccharides; Mice; Nerve Tissue Proteins; Neuralgia; Probenecid; RNA, Small Interfering; Schwann Cells
PubMed: 36195881
DOI: 10.1186/s12974-022-02603-x -
IUBMB Life Nov 2005We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA... (Review)
Review
We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA preparations. Agarose proved convenient for sizing DNA; ethidium in gel and buffer allowed visualization of DNA bands immediately after the run and improved the separation of the closed and open duplex forms of mitochondrial DNA circles. At smaller gel pore size mitochondrial DNA circles were excluded from the gel, whereas long linear DNAs were not. We concluded that the linear DNAs 'crawl like snakes head on through the gel'. This paper reviews some of the early experiments preceding the introduction of ethidium agarose gel electrophoresis.
Topics: Chemistry, Analytic; DNA; Electrophoresis, Agar Gel; Ethidium; History, 20th Century
PubMed: 16511967
DOI: 10.1080/15216540500380855 -
Scientific Reports Aug 2017DNA nanostructures represent the confluence of materials science, computer science, biology, and engineering. As functional assemblies, they are capable of performing...
DNA nanostructures represent the confluence of materials science, computer science, biology, and engineering. As functional assemblies, they are capable of performing mechanical and chemical work. In this study, we demonstrate global twisting of DNA nanorails made from two DNA origami six-helix bundles. Twisting was controlled using ethidium bromide or SYBR Green I as model intercalators. Our findings demonstrate that DNA nanorails: (i) twist when subjected to intercalators and the amount of twisting is concentration dependent, and (ii) twisting saturates at elevated concentrations. This study provides insight into how complex DNA structures undergo conformational changes when exposed to intercalators and may be of relevance when exploring how intercalating drugs interact with condensed biological structures such as chromatin and chromosomes, as well as chromatin analogous gene expression devices.
Topics: Benzothiazoles; DNA; Diamines; Ethidium; Intercalating Agents; Models, Molecular; Nanostructures; Nucleic Acid Conformation; Organic Chemicals; Quinolines
PubMed: 28785065
DOI: 10.1038/s41598-017-07796-3 -
The Journal of Biological Chemistry Dec 2017Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked...
Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in oocytes. The sensitivity toward divalent cation-mediated gating differed between small atomic ions (current) and fluorescent dye permeants, indicating that these permeants are distinctly gated. Three aspartate residues in Cx30 (Asp-50, Asp-172, and Asp-179) have been implicated previously in the Ca sensitivity of other hemichannel isoforms. Although the aspartate at position Asp-50 was indispensable for divalent cation-dependent gating of Cx30 hemichannels, substitutions of the two other residues had no significant effect on gating, illustrating differences in the gating mechanisms between connexin isoforms. Using the substituted cysteine accessibility method (SCAM), we evaluated the role of possible pore-lining residues in the permeation of ions and ethidium through Cx30 hemichannels. Of the cysteine-substituted residues, interaction of a proposed pore-lining cysteine at position 37 with the positively charged compound [2-(trimethylammonium)ethyl] methane thiosulfonate bromide (MTS-ET) increased Cx30-mediated currents with unperturbed ethidium permeability. In summary, our results demonstrate that the permeability of hemichannels is regulated in a permeant-specific manner and underscores that hemichannels are selective rather than non-discriminating and freely diffusable pores.
Topics: Amino Acid Substitution; Animals; Calcium Channels; Connexin 30; Ethidium; Gap Junctions; Humans; Ion Channel Gating; Ions; Permeability; Xenopus laevis
PubMed: 28982982
DOI: 10.1074/jbc.M117.805986 -
Memorias Do Instituto Oswaldo Cruz 1999An overview is presented of the results obtained with biodegradable sustained release devices (SRDs) containing a mixture of polymers and either isometamidium (ISMM) or... (Clinical Trial)
Clinical Trial Comparative Study Review
An overview is presented of the results obtained with biodegradable sustained release devices (SRDs) containing a mixture of polymers and either isometamidium (ISMM) or ethidium. Under controlled laboratory conditions (monthly challenge with tsetse flies infected with Trypanosoma congolense) the protection period in SRD treated cattle could be extended by a factor 2.8 (for ethidium) up to 4.2 (for ISMM) as compared to animals treated intramuscularly with the same drugs. Using a competitive drug ELISA ISMM concentrations were detected up to 330 days after the implantation of the SRDs, whereas after i.m. injection the drug was no longer present three to four months post treatment. Two field trials carried out in Mali under heavy tsetse challenge showed that the cumulative infection rate was significantly lower in the ISMM-SRD implanted cattle than in those which received ISMM intramuscularly. Using ethidium SRD, however, contradictory results were obtained in field trials in Zambia and in Mali. The potential advantages and inconvenients of the use of SRDs are discussed and suggestions are made in order to further improve the currently available devices.
Topics: Absorbable Implants; Animals; Cattle; Delayed-Action Preparations; Ethidium; Phenanthridines; Polyesters; Time Factors; Trypanocidal Agents; Trypanosoma congolense; Trypanosomiasis, African; Trypanosomiasis, Bovine
PubMed: 10224530
DOI: 10.1590/s0074-02761999000200016 -
MSphere Oct 2023Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in and other...
Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in and other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump in and other . However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility in . By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility in by directly repressing transcription of the operon, but not the other flagellum genes/operons tested. encodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to the promoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed in strains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility in to maintain homeostasis and escape hazards. IMPORTANCE Efflux and motility play a major role in bacterial growth, colonization, and survival. In , the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824-1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of the master regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress in .
Topics: Escherichia coli; Escherichia coli Proteins; Ethidium; Ligands; Anti-Bacterial Agents
PubMed: 37787551
DOI: 10.1128/msphere.00430-23 -
Current Protocols in Cell Biology Mar 2018To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and...
To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; Calibration; Cell Line; Cells; Cytological Techniques; DNA, Mitochondrial; Ethidium; Gene Dosage; Humans; Mammals; Mice; Polymerase Chain Reaction; Uracil-DNA Glycosidase; Zalcitabine
PubMed: 30040188
DOI: 10.1002/cpcb.39 -
Oxidative Medicine and Cellular... 2018Modelling of pathological processes in cells is one of the most sought-after technologies of the 21st century. Using models of such processes may help to study the... (Review)
Review
Modelling of pathological processes in cells is one of the most sought-after technologies of the 21st century. Using models of such processes may help to study the pathogenetic mechanisms of various diseases. The aim of the present study was to analyse the literature, dedicated to obtaining and investigating cybrid models. Besides, the possibility of modeling pathological processes in cells and treatment of different diseases using the models was evaluated. Methods of obtaining Rho0 cell cultures showed that, during their creation, mainly a standard technique, based on the use of mtDNA replication inhibitors (ethidium bromide), was applied. Cybrid lines were usually obtained by PEG fusion. Most frequently, platelets acted as donors of mitochondria. According to the analysis of the literature data, cybrid cell cultures can be modeled to study the dysfunction of the mitochondrial genome and molecular cellular pathological processes. Such models can be very promising for the development of therapeutic approaches to the treatment of various human diseases.
Topics: Animals; DNA, Mitochondrial; Ethidium; Genome, Mitochondrial; HEK293 Cells; Humans; Mutation
PubMed: 29983856
DOI: 10.1155/2018/4647214 -
BMC Oral Health Jan 2014There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish... (Review)
Review
BACKGROUND
There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data.
DISCUSSION
Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported.
SUMMARY
- The nomenclature regarding "viability" and "vitality" should be used carefully.- The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature.- Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.- As microbiological parameter the Plating Efficiency should be used for comparison.- Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.
Topics: Bacterial Load; Bacteriological Techniques; Biofilms; Coloring Agents; Dental Plaque; Ethidium; Fluoresceins; Fluorescence; Fluorescent Dyes; Humans; Microbial Viability; Mutagens; Terminology as Topic
PubMed: 24410850
DOI: 10.1186/1472-6831-14-2