scholarly journals Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N′-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation

1970 ◽  
Vol 116 (4) ◽  
pp. 693-707 ◽  
Author(s):  
P. D. Lawley ◽  
Carolyn J. Thatcher
Keyword(s):  
Oxygen Atom ◽  
Nucleic Acids ◽  
Mammalian Cells ◽  
Dimethyl Sulphate ◽  
Biological Effects ◽  
Methylation Of Dna ◽  
Thiol Content ◽  
Cellular Thiol ◽  
In Cells

1. In neutral aqueous solution N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O6-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects are discussed.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

scholarly journals Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

2015 ◽  
Vol 51 (37) ◽  
pp. 7887-7890 ◽  
Author(s):  
Hideto Maruyama ◽  
Kazuhiro Furukawa ◽  
Hiroyuki Kamiya ◽  
Noriaki Minakawa ◽  
Akira Matsuda
Keyword(s):  
Nucleic Acids ◽  
Mammalian Cells ◽  
Genetic Material ◽  
Rna Polymerases ◽  
Chemically Modified ◽  
Biological Studies ◽  
Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

scholarly journals Distinct Chemistries Define the Diverse Biological Effects of Plasma Activated Water Generated with Spark and Glow Plasma Discharges

2021 ◽  
Vol 11 (3) ◽  
pp. 1178
Author(s):  
Evanthia Tsoukou ◽  
Maxime Delit ◽  
Louise Treint ◽  
Paula Bourke ◽  
Daniela Boehm
Keyword(s):  
Mammalian Cells ◽  
Biomedical Application ◽  
Biological Effects ◽  
Mode Of Delivery ◽  
Chemical Species ◽  
Resistant Bacteria ◽  
Cytotoxic Activities ◽  
Promising Alternative ◽  
Plasma Activated Water

The spread of multidrug-resistant bacteria poses a significant threat to human health. Plasma activated liquids (PAL) could be a promising alternative for microbial decontamination, where different PAL can possess diverse antimicrobial efficacies and cytotoxic profiles, depending on the range and concentration of their reactive chemical species. In this research, the biological activity of plasma activated water (PAW) on different biological targets including both microbiological and mammalian cells was investigated in vitro. The aim was to further an understanding of the specific role of distinct plasma reactive species, which is required to tailor plasma activated liquids for use in applications where high antimicrobial activity is required without adversely affecting the biology of eukaryotic cells. PAW was generated by glow and spark discharges, which provide selective generation of hydrogen peroxide, nitrite and nitrate in the liquid. The PAW made by either spark or glow discharges showed similar antimicrobial efficacy and stability of activity, despite the very different reactive oxygen species (ROS) and reactive nitrogen species profiles (RNS). However, different trends were observed for cytotoxic activities and effects on enzyme function, which were translated through the selective chemical species generation. These findings indicate very distinct mechanisms of action which may be exploited when tailoring plasma activated liquids to various applications. A remarkable stability to heat and pressure was noted for PAW generated with this set up, which broadens the application potential. These features also suggest that post plasma modifications and post generation stability can be harnessed as a further means of modulating the chemistry, activity and mode of delivery of plasma functionalised liquids. Overall, these results further understanding on how PAL generation may be tuned to provide candidate disinfectant agents for biomedical application or for bio-decontamination in diverse areas.

scholarly journals IN VITRO ASSESSMENT OF THE BIOLOGICAL ACTIVITY OF A NEW REGENERATIVE AGENT PREPARED FROM THE CONCENTRATE OF DEPROTEINIZED DERMAL LAYER OF PORCINE SKIN

2020 ◽  
pp. 12-22
Author(s):  
Natalia Bezdieniezhnykh ◽  
Aleksandra Lykhova ◽  
Hennadii Borschevskyi ◽  
Kateryna Dyakun ◽  
Ievgen Kruglov
Keyword(s):  
Biological Activity ◽  
Cell Line ◽  
Mammalian Cells ◽  
Therapeutic Agent ◽  
Biological Effects ◽  
Mdbk Cell ◽  
Porcine Skin ◽  
Bovine Kidney ◽  
Dermal Layer

Background. Presently, a prospective direction for the development of regenerative medicine in the world is the search for regulatory molecules and the identification of molecular targets to stimulate the body's endogenous regenerative potential. The concentrate of the deproteinized dermal layer of porcine skin (СDDLPS) is a new therapeutic agent with restorative properties, the action of which is directed on the induction of the self resources of cells. Aim. The assessment of the effect of СDDLPS on the proliferative activity of mammalian cells of different histogenesis in vitro. Materials and Methods. To determine the amino acid composition of the СDDLPS liquid chromatography and biochemical methods were used. The biological effects and mechanisms of action of the drug were investigated by cell culture and molecular biological methods. The research was carried out using stable cell lines: human keratinocytes (HaCaT cell line), porcine endothelial cells (PAE cell line), bovine kidney cells (MDBK cell line) and mouse fibroblasts (3T3A31 cell line). Results. The cells of the bovine kidney MDBK cell line were the most sensitive to the effect of the CDDLPS. Also, the obtained results suggest that, depending on the concentration, the drug not only stimulates cell proliferation by 10–30 %, but also significantly enhances biosynthetic processes in cells, in particular, protein synthesis by 20–40 %. Conclusions. CDDLPS is an effective and affordable therapeutic agent with restorative properties, the biological activity of which manifests itself in the activation of cell biosynthetic and proliferative potentials and is comparable to effects of some growth factors, in particular epidermal growth factor

scholarly journals Oxidative Potential Versus Biological Effects: A Review on the Relevance of Cell-Free/Abiotic Assays as Predictors of Toxicity from Airborne Particulate Matter

2019 ◽  
Vol 20 (19) ◽  
pp. 4772 ◽  
Author(s):  
Johan Øvrevik
Keyword(s):  
Particulate Matter ◽  
Biological Effects ◽  
Positive Association ◽  
Ambient Air ◽  
Epidemiological Studies ◽  
In Vivo Studies ◽  
Oxidative Potential ◽  
In Cells

Background and Objectives: The oxidative potential (OP) of particulate matter (PM) in cell-free/abiotic systems have been suggested as a possible measure of their biological reactivity and a relevant exposure metric for ambient air PM in epidemiological studies. The present review examined whether the OP of particles correlate with their biological effects, to determine the relevance of these cell-free assays as predictors of particle toxicity. Methods: PubMed, Google Scholar and Web of Science databases were searched to identify relevant studies published up to May 2019. The main inclusion criteria used for the selection of studies were that they should contain (1) multiple PM types or samples, (2) assessment of oxidative potential in cell-free systems and (3) assessment of biological effects in cells, animals or humans. Results: In total, 50 independent studies were identified assessing both OP and biological effects of ambient air PM or combustion particles such as diesel exhaust and wood smoke particles: 32 in vitro or in vivo studies exploring effects in cells or animals, and 18 clinical or epidemiological studies exploring effects in humans. Of these, 29 studies assessed the association between OP and biological effects by statistical analysis: 10 studies reported that at least one OP measure was statistically significantly associated with all endpoints examined, 12 studies reported that at least one OP measure was significantly associated with at least one effect outcome, while seven studies reported no significant correlation/association between any OP measures and any biological effects. The overall assessment revealed considerable variability in reported association between individual OP assays and specific outcomes, but evidence of positive association between intracellular ROS, oxidative damage and antioxidant response in vitro, and between OP assessed by the dithiothreitol (DDT) assay and asthma/wheeze in humans. There was little support for consistent association between OP and any other outcome assessed, either due to repeated lack of statistical association, variability in reported findings or limited numbers of available studies. Conclusions: Current assays for OP in cell-free/abiotic systems appear to have limited value in predicting PM toxicity. Clarifying the underlying causes may be important for further advancement in the field.

scholarly journals Association of Ebola Virus Matrix Protein VP40 with Microtubules

2005 ◽  
Vol 79 (8) ◽  
pp. 4709-4719 ◽  
Author(s):  
Gordon Ruthel ◽  
Gretchen L. Demmin ◽  
George Kallstrom ◽  
Melodi P. Javid ◽  
Shirin S. Badie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Life Cycles ◽  
Binding Motif ◽  
Physical Interaction ◽  
Tubulin Polymerization ◽  
In Cells

ABSTRACT Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

scholarly journals ICBP90, a Novel Methyl K9 H3 Binding Protein Linking Protein Ubiquitination with Heterochromatin Formation

2007 ◽  
Vol 28 (2) ◽  
pp. 705-717 ◽  
Author(s):  
Panagiota Karagianni ◽  
Larbi Amazit ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Binding Protein ◽  
Biological Effects ◽  
Binding Activity ◽  
Effector Proteins ◽  
Heterochromatin Formation ◽  
Protein Ubiquitination

ABSTRACT Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

Genetic and Cellular Toxicology of Dental Resin Monomers

2006 ◽  
Vol 85 (10) ◽  
pp. 870-877 ◽  
Author(s):  
H. Schweikl ◽  
G. Spagnuolo ◽  
G. Schmalz
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Triethylene Glycol ◽  
Redox Balance ◽  
Radical Scavenger ◽  
Dental Resin ◽  
Vast Number ◽  
New Findings

Monomers are released from dental resin materials, and thus cause adverse biological effects in mammalian cells. Cytotoxicity and genotoxicity of some of these methacrylates have been identified in a vast number of investigations during the last decade. It has been well-established that the co-monomer triethylene glycol dimethacrylate (TEGDMA) causes gene mutations in vitro. The formation of micronuclei is indicative of chromosomal damage and the induction of DNA strand breaks detected with monomers like TEGDMA and 2-hydroxyethyl methacrylate (HEMA). As a consequence of DNA damage, the mammalian cell cycle was delayed in both G1 and G2/M phases, depending on the concentrations of the monomers. Yet, the mechanisms underlying the genetic and cellular toxicology of resin monomers have remained obscure until recently. New findings indicate that increased oxidative stress results in an impairment of the cellular pro- and anti-oxidant redox balance caused by monomers. It has been demonstrated that monomers reduced the levels of the natural radical scavenger glutathione (GSH), which protects cell structures from damage caused by reactive oxygen species (ROS). Depletion of the intracellular GSH pool may then significantly contribute to cytotoxicity, because a related increase in ROS levels can activate pathways leading to apoptosis. Complementary, cytotoxic, and genotoxic effects of TEGDMA and HEMA are inhibited in the presence of ROS scavengers like N-acetylcysteine (NAC), ascorbate, and Trolox (vitamin E). Elevated intracellular levels of ROS can also activate a complex network of redox-responsive macromolecules, including redox-sensitive transcription factors like nuclear factor kappaB (NF-κB). It has been shown that NF-κB is activated probably to counteract HEMA-induced apoptosis. The induction of apoptosis by TEGDMA in human pulp cells has been associated with an inhibition of the phosphatidylinositol 3-kinase (PI3-K) cell-survival signaling pathway. Although the details of the mechanisms leading to cell death, genotoxicity, and cell-cycle delay are not completely understood, resin monomers may be able to alter the functions of the cells of the oral cavity. Pathways regulating cellular homeostasis, dentinogenesis, or tissue repair may be modified by monomers at concentrations well below those which cause acute cytotoxicity.

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