cis—Dichlorodiammineplatinum (II) and its Effect on Microfilaments in Mammalian Cells

1975 ◽  
Vol 33 ◽  
pp. 394-395
Author(s):  
S.K. Aggarwal ◽  
P. McAllister
Keyword(s):  
Immune System ◽  
Dimethyl Sulfoxide ◽  
Cell Cultures ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Fresh Medium ◽  
Sarcoma 180 ◽  
Heavy Meromyosin ◽  
Cells In Culture

The regression of various tumors after cis-dichlorodiammineplatinum (II) (cis-Pt (II) treatment has been suggested to be mediated through mitotic inhibition and enhancement of the immune system. Present study illustrates the possible action of cis-Pt (II) on microfilaments and their role in the prevention of cytokinesis.Sarcoma-180 ascites cells in culture were treated with cis-Pt(II) (5ppm; 2 μg/ml) for intervals of 15, 30, 60 and 120 minutes. Controls were incubated in culture medium without cis-Pt (II) . Both the treated and non-treated cultures were then glycerinated and incubated with heavy meromyosin (HMM) (Ishikawa, et al., 1969. J. Cell Biol., 43, 312). Heavy meromyosin was prepared from myosin (Lowery, et al., 1969, J. Mol. Biol., 42, 1) using trypsin as inhibitor. Cell cultures treated with cis-Pt(II) for 30 minutes were also transferred to fresh medium containing 25μg/ml cytochalasin B and 1% dimethyl sulfoxide (DMSO) for 2-24 hours.

scholarly journals Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

1984 ◽  
Vol 4 (11) ◽  
pp. 2449-2454 ◽  
Author(s):  
E R Kaufman
Keyword(s):  
Syrian Hamster ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Cells In Culture ◽  
Thymidine Analog ◽  
High Concentrations

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells

1984 ◽  
Vol 4 (11) ◽  
pp. 2449-2454
Author(s):  
E R Kaufman
Keyword(s):  
Syrian Hamster ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Cells In Culture ◽  
Thymidine Analog ◽  
High Concentrations

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.

scholarly journals The stability of envelope-pseudotyped lentiviral vectors

Gene Therapy ◽  
2020 ◽  
Author(s):  
Iris J. C. Dautzenberg ◽  
Martijn J. W. E. Rabelink ◽  
Rob C. Hoeben
Keyword(s):  
Cell Cultures ◽  
Half Life ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Lentiviral Vector ◽  
Virus Titer ◽  
Lentiviral Vectors ◽  
Envelope Proteins ◽  
The Stability ◽  
Vector Particles

Abstract Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the ‘Reduction Ratio.’ This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector’s envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2–110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.

scholarly journals Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

1985 ◽  
Vol 5 (11) ◽  
pp. 3092-3096 ◽  
Author(s):  
E R Kaufman
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Chemical Mutagen ◽  
Genetic Studies ◽  
Cells In Culture ◽  
Thymidine Analog ◽  
High Concentrations ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
High Degree ◽  
Spontaneous Mutants

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells

1985 ◽  
Vol 5 (11) ◽  
pp. 3092-3096
Author(s):  
E R Kaufman
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Chemical Mutagen ◽  
Genetic Studies ◽  
Cells In Culture ◽  
Thymidine Analog ◽  
High Concentrations ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
High Degree ◽  
Spontaneous Mutants

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Effect of pulsing electromagnetic fields on DNA synthesis in mammalian cells in culture

1986 ◽  
Vol 42 (2) ◽  
pp. 185-186 ◽  
Author(s):  
K. Takahashi ◽  
I. Kaneko ◽  
M. Date ◽  
E. Fukada
Keyword(s):  
Dna Synthesis ◽  
Electromagnetic Fields ◽  
Mammalian Cells ◽  
Cells In Culture
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